Methods and compositions for treating nafld, hepatic steatosis, and sequelae thereof

ABSTRACT

Provided herein are oral pharmaceutical compositions containing a GLP-1 analogue and/or insulin for treating and reducing the incidence of nonalcoholic fatty liver disease (NAFLD), hepatic steatosis, and sequelae thereof and methods of utilizing same.

FIELD

Provided herein are oral pharmaceutical compositions containing a GLP-1analogue and/or insulin for treating and reducing the incidence ofnonalcoholic fatty liver disease (NAFLD), hepatic steatosis, andsequelae thereof.

BACKGROUND

The incretin hormone Glucagon-like Peptide 1 (GLP-1), secreted withinminutes of food ingestion, is associated with induction of insulinrelease. GLP-1 is used in therapies for Type 2 Diabetes Mellitus (T2DM).

Clinical use of the native GLP-1 is limited due to its rapid enzymaticinactivation, resulting in a half-life of 2-3 minutes. To overcome thisobstacle, long-acting, degradation-resistant peptides, both natural andsynthetic, referred to as GLP-1 mimetic agents or analogues, have beendesigned and are being used.

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of chronicdiseases including fatty liver, or bland steatosis, and sequelae ofbland steatosis, such as non-alcoholic steatohepatitis (NASH), lobularnecroinflammation with fibrosis, and cirrhosis (Sharma et al, 2011; Puriet al, 2008). NAFLD-related cirrhosis can lead to end-stage liverdisease and hepatocellular carcinoma (HCC). Like other chronic end-stageliver diseases, the major option for afflicted persons is livertransplantation. NAFLD appears to be associated with certaindyslipidemias (Malhi et al, 2008; Retnakaran R et al 2012; Sung et al,2012). An important component of Western diets includes both saturatedfatty acids as well as trans-saturated fatty acids. Recent evidenceindicates that such fatty acids are partly responsible for inducingsteatosis and fueling hepatocyte insulin resistance (Centis et al,2010).

Some studies have suggested that GLP-1 reduces fat load in hepatocytesand may be useful in treating NAFLD, although other studies havedemonstrated that GLP-1 increases hepatic glycogen synthase activity,which is expected to aggravate NAFLD. Indeed, even the very presence ofGLP-1 receptor in liver cells remains controversial (Nielsen et al 2008,Redondo et al, 2003; Lopez-Delgado et al, 1998; Sharma et al, 2011;Ben-Shlomo et al 2011). A more rigorous assessment of inflammation andfibrosis in response to GLP-1 is considered necessary in order to morefully understand the response of liver cells to these agents (Kim et al,2012).

Alzheimer's disease (AD), also known as Alzheimer disease, is the mostcommon form of dementia. There is no cure for the disease, which worsensas it progresses and eventually leads to death. Most often, AD isdiagnosed in people over 65 years of age, although the less-prevalentearly-onset Alzheimer's can occur much earlier. In 2006, there were 26.6million sufferers worldwide. The causation of AD is not well understood,although it is typically correlated with accumulation of amyloid βpeptides and tau protein in the brain, and these substances are believedby many scientists to play a role in causation.

Parkinson's disease (PD; also known as idiopathic or primaryparkinsonism, hypokinetic rigid syndrome/HRS, or paralysis agitans) is adegenerative disorder of the central nervous system. The motor symptomsof Parkinson's disease result from the death of dopamine-generatingcells in the substantia nigra, a region of the midbrain, the cause ofwhich is unknown. The disease course typically presents withmovement-related symptoms, with thinking and behavioral problems arisinglater and dementia occurring in advanced disease. Parkinson's disease ismore common in older people, with most cases occurring after the age of50.

Huntington's disease (HD) is a neurodegenerative genetic disorder thataffects muscle coordination and leads to cognitive decline andpsychiatric problems. It typically becomes noticeable in mid-adult life.The huntingtin gene provides the genetic information for a protein thatis also called “Huntingtin”. Expansion of a CAG triplet repeat stretchwithin huntingtin results in a mutant form of the protein, whichgradually damages cells in the brain, through mechanisms that are notfully understood.

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's diseaseand motor neuron disease, is an incurable neurodegenerative disorder ofthe voluntary motor system. Characterized by selective and progressivedeath of motor neurons within the brain and spinal cord, it leads toparalysis of voluntary muscles and, eventually, death within five yearsof clinical onset. Most cases of ALS occur sporadically with unknownetiology (Li et al).

Traumatic brain injury (TBI) affects 1.7 million Americans each year andis a primary contributing factor (30.5%) of all injury-related deaths inthe United States. The elderly, in particular, are vulnerable to TBI andsuffer an increased mortality and worse functional outcome in the faceof lower initial injury severity. Many survivors experience prolonged oreven permanent neurocognitive dysfunction, with lasting changes incognition, motor function, and personality (Eakin et al).

Mood disorders (e.g. bipolar disorder (BD) and major depressive disorder(MDD)) are highly prevalent, affecting 10-15% of the population at sometime in their life. Mood disorders are also associated with a range ofcognitive deficits which often persist during euthymic states in bothtreated and untreated individuals. Cognitive deficits are documentedacross most domains of function, including but not limited to, deficitsin learning, memory (e.g., working memory, episodic memory, and semanticmemory), attention, executive function, processing speed, and socialcognition. However, conventional pharmacological agents for mooddisorders are not proven to be sufficiently effective in treatingobjectively and/or subjectively measured cognitive deficits (McIntyre etal).

Cerebral stroke is caused by blockage or rupture of a blood vessel,which causes a disruption in blood flow to the surrounding tissues. Thetissues in the area of the stroke can become irreversibly damaged andultimately become necrotic and die. While this cell death and resultingbrain damage may not be preventable, there is a region of tissue thatmay be amenable to therapeutic manipulation in the area surrounding thedamaged stroke infarct zone (Salcedo et al).

Diabetic retinopathy, one form of retinal degeneration, is damage to theretina caused by complications of diabetes, which can eventually lead toblindness. It is an ocular manifestation of diabetes, a systemicdisease, which affects up to 80 percent of all patients who have haddiabetes for 10 years or more (Kertes et al).

Peripheral neuropathy (PN) is damage or disease of nerves of theperipheral nervous system. It may affect sensation, movement, gland ororgan function, and other aspects of health, depending on the type ofnerve affected. PN may be caused by systemic diseases such as diabetesor leprosy, vitamin deficiency, medication (e.g., chemotherapy),traumatic injury, excessive alcohol consumption, immune system disease,or infection, or may be inherited (Hughes).

To date, GLP-1 analogues are only available as injectable dosage forms.The present inventors are developing an oral exenatide (a GLP-1analogue) formulation for use in treating diabetes. A first-in-humanstrial (n=4) testing its safety in healthy humans demonstrated biologicalfunctionality of orally delivered exenatide (Eldor et al 2010).

SUMMARY

Provided herein are oral pharmaceutical compositions containing a GLP-1analogue and/or insulin for treating and preventing nonalcoholic fattyliver disease (NAFLD), hepatic steatosis, and sequelae thereof, andmethods of utilizing same. Results from subcutaneous, systemic, andother routes of administration cannot be extrapolated to oraladministration, since oral dosage forms tend to utilize the portal routeof absorption. To the knowledge of the inventors, orally administeredGLP-1 analogues have never been tested for treatment of NAFLD, sequelaethereof, or neurodegenerative disease.

It is described herein that the described compositions are capable oftreating and preventing sequelae of NAFLD, as well as other therapeuticeffects mediated by GLP-1 receptors outside the pancreas. Accordingly,the described compositions are capable of treating and preventingobesity, elevated total cholesterol, hypertriglyceridemia, elevatedserum ApoB levels, elevated total cholesterol/HDL ratios, and elevatedApoB/ApoA1 ratios, atherosclerosis, sub-clinical inflammation, aprothrombotic state, platelet activation, endothelial dysfunction, acardioembolic state, and impaired insulin-induced enhancement ofvasodilator responses (Sung et al, 2012; Chatrath et al 2012; Nseir etal, 2011; Salcedo et al, 2012). It is also described that the describedcompositions are capable of treating various neurodegenerativedisorders.

Also provided herein are solid pharmaceutical compositions foradministration of a therapeutic protein to a subject.

The terms “protein” and “peptide” are used interchangeably herein.Neither term is intended to confer a limitation of the number of aminoacids present, except where a limitation is explicitly indicated.

Unless indicated otherwise, all ranges mentioned herein are inclusive.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Provided herein is a pharmaceutical composition for treating or reducingthe incidence of nonalcoholic fatty liver disease (NAFLD) in a human,said pharmaceutical composition comprising a GLP-1 analogue, at leastone protease inhibitor, and a chelator of divalent cations. In anotherembodiment, the pharmaceutical composition comprises a GLP-1 analogue,insulin, at least one protease inhibitor, and a chelator of divalentcations. In still other embodiments, the pharmaceutical compositioncomprises insulin, at least one protease inhibitor, and a chelator ofdivalent cations. In certain embodiments, the pharmaceutical compositionis administered to the subject for an extended time.

In some embodiments, the pharmaceutical compositions described hereinare solid formulations. In other embodiments, the describedpharmaceutical compositions comprise a liquid formulation, wherein saidformulation is, in certain embodiments, surrounded by a capsule and/or acoating that resists degradation in the stomach. In some embodiments,the liquid formulation is an oil-based liquid formulation. Someembodiments of the liquid formulation comprise a GLP-1 analogue,insulin, at least one protease inhibitor, examples of which are trypsininhibitors and chymotrypsin inhibitors, and a chelator of divalentcations. In other embodiments, the liquid formulation comprises a GLP-1analogue, at least one protease inhibitor, and a chelator of divalentcations. In other embodiments, the liquid formulation comprises insulin,at least one protease inhibitor, and a chelator of divalent cations.Each described embodiment regarding the GLP-1 analogue and amountthereof, insulin and amount thereof, protease inhibitors, chelators,coatings, etc, is also intended to be considered a specific embodimentin the context of liquid formulations in general and oil-based liquidformulations in particular and also in the context of the solidformulations described herein. Those skilled in the art will appreciate,in light of the present disclosure, that solid dosage forms may comprisea different list of inactive excipients from the described liquidformulations.

In some embodiments, the subject receiving the described pharmaceuticalcomposition already has NAFLD. In other embodiments, the subject is atrisk of developing NAFLD.

In yet another aspect, a use of a combination of ingredients describedherein is provided in the preparation of a medicament for treating NAFLDin a human.

Still another aspect provides a method for treating or reducing theincidence of NAFLD in a human, the method comprising the optional stepof selecting a subject by diagnosing NAFLD, followed by the step ofadministering to a subject in need of such treatment a pharmaceuticalcomposition described herein, thereby treating or reducing the incidenceof NAFLD in a human.

In certain embodiments, the treated disorder is selected from the groupconsisting of bland steatosis, non-alcoholic steatohepatitis (NASH), andlobular necroinflammation with fibrosis. In other embodiments, thedisorder is bland steatosis; in other embodiments, it is non-alcoholicsteatohepatitis (NASH); and in still other embodiments, it is lobularnecroinflammation with fibrosis.

Those skilled in the art will appreciate in light of the presentdisclosure that NAFLD and sequelae thereof may be present in subjectswith diabetes, subjects exhibiting insulin resistance who do not havefrank diabetes, and subjects that do not exhibit insulin resistance. Insome embodiments, the subject treated by the described methods andcompositions exhibits insulin resistance but does not have frankdiabetes. In other embodiments, the subject does not exhibit insulinresistance. In other embodiments, the subject is obese but haswell-controlled plasma glucose levels. In other embodiments, the treatedsubject is non-obese (Younossi et al 2012).

Methods of diagnosing and measuring non-alcoholic steatohepatitis arewell known in the art, and include, for example, ultrasound, for examplehepatorenal echo contrast, liver brightness, and vascular blurring underabdominal ultrasonography (Saverymuttu et al); measurement of liverenzyme levels, e.g. aspartate transaminase (AST), alanine transaminase(ALT), alkaline phosphatase, and gamma-glutamyl transpeptidase (GGT);hepatic mRNA levels of genes involved in lipogenesis; and hepaticdiacylglycerol acyltransferase-2 (DGAT2) levels (Miyashita T et al 2012;Sung et al 2012; and Juurinen et al 2007); magnetic resonance imaging(Mazhar et al, 2009); and measuring adiponectin levels.

It will be appreciated by those skilled in the art that variousdisorders disclosed herein are sequelae of NAFLD (see, for example, Sunget al, 2012; Chatrath et al 2012; Nseir et al, 2011; Salcedo et al,2012). In other cases, as will also be appreciated by those skilled inthe art, GLP-1 can have a therapeutic effect independently of itseffects on NAFLD.

Provided herein, in another embodiment, is a pharmaceutical compositionfor treating, or in another aspect reducing the incidence of, elevatedtotal cholesterol in a human with hepatic steatosis, said pharmaceuticalcomposition comprising insulin, a GLP-1 analogue, at least one proteaseinhibitor, and a chelator of divalent cations. In another embodiment,the pharmaceutical composition comprises a GLP-1 analogue, at least oneprotease inhibitor, and a chelator of divalent cations. In yet anotherembodiment, the pharmaceutical composition comprises insulin, at leastone protease inhibitor, and a chelator of divalent cations. In certainembodiments, the pharmaceutical composition comprises one of theoil-based liquid formulations described herein, wherein thepharmaceutical composition may further comprise, in some embodiments, acapsule and/or coating that resists degradation in the stomach. In otherembodiments, the pharmaceutical composition comprises one of the solidformulations described herein. In certain embodiments, thepharmaceutical composition is administered to the subject for anextended time.

In yet another aspect, a use of a combination of ingredients describedherein is provided in the preparation of a medicament for treating, orin another aspect reducing the incidence of, elevated total cholesterolin a human with hepatic steatosis.

Still another aspect provides a method of treating, or in another aspectreducing the incidence of, elevated total cholesterol in a subject withhepatic steatosis, the method comprising the step of administering to asubject with hepatic steatosis a pharmaceutical composition describedhereinabove, thereby treating or reducing the incidence of elevatedtotal cholesterol.

Provided herein, in another embodiment, is a pharmaceutical compositionfor treating, or in another aspect reducing the incidence of,hypertriglyceridemia in a human with hepatic steatosis, saidpharmaceutical composition comprising an insulin, a GLP-1 analogue, atleast one protease inhibitor, and a chelator of divalent cations. Inanother embodiment, the pharmaceutical composition comprises a GLP-1analogue, at least one protease inhibitor, and a chelator of divalentcations. In yet another embodiment, the pharmaceutical compositioncomprises insulin, at least one protease inhibitor, and a chelator ofdivalent cations. In certain embodiments, the pharmaceutical compositioncomprises one of the oil-based liquid formulations described herein,wherein the pharmaceutical composition may further comprise, in someembodiments, a capsule and/or coating that resists degradation in thestomach. In other embodiments, the pharmaceutical composition comprisesone of the solid formulations described herein. In certain embodiments,the pharmaceutical composition is administered to the subject for anextended time.

In yet another aspect, a use of a combination of ingredients describedherein is provided in the preparation of a medicament for treating, orin another aspect reducing the incidence of, hypertriglyceridemia in ahuman with hepatic steatosis.

Still another aspect provides a method of treating, or in another aspectreducing the incidence of, hypertriglyceridemia in a subject withhepatic steatosis, the method comprising the step of administering to asubject with hepatic steatosis a pharmaceutical composition describedhereinabove, thereby treating or reducing the incidence ofhypertriglyceridemia.

Provided herein, in another embodiment, is a pharmaceutical compositionfor treating, or in another aspect reducing the incidence of, anelevated serum apolipoprotein B (ApoB) level in a human with hepaticsteatosis, said pharmaceutical composition comprising insulin, a GLP-1analogue, at least one protease inhibitor, and a chelator of divalentcations. In another embodiment, the pharmaceutical composition comprisesa GLP-1 analogue, at least one protease inhibitor, and a chelator ofdivalent cations. In yet another embodiment, the pharmaceuticalcomposition comprises insulin, at least one protease inhibitor, and achelator of divalent cations. In certain embodiments, the pharmaceuticalcomposition comprises one of the oil-based liquid formulations describedherein, wherein the pharmaceutical composition may further comprise, insome embodiments, a capsule and/or coating that resists degradation inthe stomach. In other embodiments, the pharmaceutical compositioncomprises one of the solid formulations described herein. In certainembodiments, the pharmaceutical composition is administered to thesubject for an extended time.

In yet another aspect, a use of a combination of ingredients describedherein is provided in the preparation of a medicament for treating, orin another aspect reducing the incidence of, an elevated serum ApoBlevel in a human with hepatic steatosis.

Still another aspect provides a method of treating, or in another aspectreducing the incidence of, an elevated serum ApoB level in a subjectwith hepatic steatosis, the method comprising the step of administeringto a subject with hepatic steatosis a pharmaceutical compositiondescribed hereinabove, thereby treating or reducing the incidence ofelevated serum ApoB.

Provided herein, in another embodiment, is a pharmaceutical compositionfor treating, or in another aspect reducing the incidence of, anelevated total cholesterol/HDL ratio in a human with hepatic steatosis,said pharmaceutical composition comprising insulin, a GLP-1 analogue, atleast one protease inhibitor, and a chelator of divalent cations. Inanother embodiment, the pharmaceutical composition comprises a GLP-1analogue, at least one protease inhibitor, and a chelator of divalentcations. In yet another embodiment, the pharmaceutical compositioncomprises insulin, at least one protease inhibitor, and a chelator ofdivalent cations. In certain embodiments, the pharmaceutical compositioncomprises one of the oil-based liquid formulations described herein,wherein the pharmaceutical composition may further comprise, in someembodiments, a capsule and/or coating that resists degradation in thestomach. In other embodiments, the pharmaceutical composition comprisesone of the solid formulations described herein. In certain embodiments,the pharmaceutical composition is administered to the subject for anextended time.

In yet another aspect, a use of a combination of ingredients describedherein is provided in the preparation of a medicament for treating, orin another aspect reducing the incidence of, an elevated totalcholesterol/HDL ratio in a human with hepatic steatosis.

Still another aspect provides a method of treating, or in another aspectreducing the incidence of, an elevated total cholesterol/HDL ratio in asubject with hepatic steatosis, the method comprising the step ofadministering to a subject with hepatic steatosis a pharmaceuticalcomposition described hereinabove, thereby treating or reducing theincidence of an elevated total cholesterol/HDL ratio.

Provided herein, in another embodiment, is a pharmaceutical compositionfor treating, or in another aspect reducing the incidence of, anelevated apolipoprotein B (ApoB)/apolipoprotein A1 (ApoA1) ratio in ahuman with hepatic steatosis, said pharmaceutical composition comprisinginsulin, a GLP-1 analogue, at least one protease inhibitor, and achelator of divalent cations. In another embodiment, the pharmaceuticalcomposition comprises a GLP-1 analogue, at least one protease inhibitor,and a chelator of divalent cations. In yet another embodiment, thepharmaceutical composition comprises insulin, at least one proteaseinhibitor, and a chelator of divalent cations. In certain embodiments,the pharmaceutical composition comprises one of the oil-based liquidformulations described herein, wherein the pharmaceutical compositionmay further comprise, in some embodiments, a capsule and/or coating thatresists degradation in the stomach. In other embodiments, thepharmaceutical composition comprises one of the solid formulationsdescribed herein. In certain embodiments, the pharmaceutical compositionis administered to the subject for an extended time.

In yet another aspect, a use of a combination of ingredients describedherein is provided in the preparation of a medicament for treating, orin another aspect reducing the incidence of, an elevated ApoB/ApoA1ratio in a human with hepatic steatosis.

Still another aspect provides a method of treating, or in another aspectreducing the incidence of, an elevated ApoB/ApoA 1 ratio in a subjectwith hepatic steatosis, the method comprising the step of administeringto a subject with hepatic steatosis a pharmaceutical compositiondescribed hereinabove, thereby treating or reducing the incidence of anelevated ApoB/ApoA1 ratio.

Methods for measuring each of the aforementioned lipid parameters arewell known to those skilled in the art. Exemplary methods are describedinter alia in Chiquette E et al and Martinez-Colubi M et al.

Provided herein, in another embodiment, is a pharmaceutical compositionfor treating, or in another aspect reducing the incidence of, animpaired insulin-induced enhancement of vasodilator response in asubject, said pharmaceutical composition comprising insulin, a GLP-1analogue, at least one protease inhibitor, and a chelator of divalentcations. In another embodiment, the pharmaceutical composition comprisesa GLP-1 analogue, at least one protease inhibitor, and a chelator ofdivalent cations. In yet another embodiment, the pharmaceuticalcomposition comprises insulin, at least one protease inhibitor, and achelator of divalent cations. In certain embodiments, the pharmaceuticalcomposition comprises one of the oil-based liquid formulations describedherein, wherein the pharmaceutical composition may further comprise, insome embodiments, a capsule and/or coating that resists degradation inthe stomach. In other embodiments, the pharmaceutical compositioncomprises one of the solid formulations described herein. In certainembodiments, the pharmaceutical composition is administered to thesubject for an extended time.

In yet another aspect, a use of a combination of ingredients describedherein is provided in the preparation of a medicament for treating, orin another aspect reducing the incidence of, an impaired insulin-inducedenhancement of vasodilator response in a subject.

Still another aspect provides a method of treating, or in another aspectreducing the incidence of, an impaired insulin-induced enhancement ofvasodilator response in a subject, the method comprising the step ofadministering to a subject a pharmaceutical composition describedhereinabove, thereby treating or reducing the incidence of an impairedinsulin-induced enhancement of vasodilator response.

Another aspect provides a method of treating an impaired insulin-inducedenhancement of vasodilator responses in a subject with NAFLD, the methodcomprising the step of administering to a subject in need of suchtreatment a pharmaceutical composition described hereinabove, therebytreating an impaired insulin-induced enhancement of vasodilatorresponses in a subject with NAFLD. Methods for measuring insulin-inducedvasodilator responses are known in the art, and include, for example,measuring blood flow responses (for example in the forearm) toacetylcholine (ACh) and sodium nitroprusside (SNP) (Tesauro et al).

Provided herein, in another embodiment, is a pharmaceutical compositionfor reducing, or in another aspect reducing the incidence of, fatdeposition in the liver, said pharmaceutical composition comprisinginsulin, a GLP-1 analogue, at least one protease inhibitor, and achelator of divalent cations. In another embodiment, the pharmaceuticalcomposition comprises a GLP-1 analogue, at least one protease inhibitor,and a chelator of divalent cations. In yet another embodiment, thepharmaceutical composition comprises insulin, at least one proteaseinhibitor, and a chelator of divalent cations. In certain embodiments,the pharmaceutical composition comprises one of the oil-based liquidformulations described herein, wherein the pharmaceutical compositionmay further comprise, in some embodiments, a capsule and/or coating thatresists degradation in the stomach. In other embodiments, thepharmaceutical composition comprises one of the solid formulationsdescribed herein. In certain embodiments, the pharmaceutical compositionis administered to the subject for an extended time.

In yet another aspect, a use of a combination of ingredients describedherein is provided in the preparation of a medicament for reducing, orin another aspect reducing the incidence of, fat deposition in theliver.

Still another aspect provides a method of reducing, or in another aspectreducing the incidence of, fat deposition in the liver, the methodcomprising the step of administering to a subject a pharmaceuticalcomposition described hereinabove, thereby reducing or reducing theincidence of fat deposition in the liver.

Provided herein, in another embodiment, is a pharmaceutical compositionfor preventing or reducing the incidence of a cardioembolism, saidpharmaceutical composition comprising insulin, a GLP-1 analogue, atleast one protease inhibitor, and a chelator of divalent cations. Inanother embodiment, the pharmaceutical composition comprises a GLP-1analogue, at least one protease inhibitor, and a chelator of divalentcations. In yet another embodiment, the pharmaceutical compositioncomprises insulin, at least one protease inhibitor, and a chelator ofdivalent cations. In certain embodiments, the pharmaceutical compositioncomprises one of the oil-based liquid formulations described herein,wherein the pharmaceutical composition may further comprise, in someembodiments, a capsule and/or coating that resists degradation in thestomach. In other embodiments, the pharmaceutical composition comprisesone of the solid formulations described herein. In certain embodiments,the pharmaceutical composition is administered to the subject for anextended time.

In yet another aspect, a use of a combination of ingredients describedherein is provided in the preparation of a medicament for preventing orreducing the incidence of a cardioembolism.

Still another aspect provides a method of preventing or reducing theincidence of a cardioembolism, the method comprising the step ofadministering to a subject a pharmaceutical composition describedhereinabove, thereby preventing or reducing the incidence of acardioembolism.

Provided herein, in another embodiment, is a pharmaceutical compositionfor reversing, or in another aspect reducing the incidence of,endothelial dysfunction, said pharmaceutical composition comprisinginsulin, a GLP-1 analogue, at least one protease inhibitor, and achelator of divalent cations. In another embodiment, the pharmaceuticalcomposition comprises a GLP-1 analogue, at least one protease inhibitor,and a chelator of divalent cations. In yet another embodiment, thepharmaceutical composition comprises insulin, at least one proteaseinhibitor, and a chelator of divalent cations. In certain embodiments,the pharmaceutical composition comprises one of the oil-based liquidformulations described herein, wherein the pharmaceutical compositionmay further comprise, in some embodiments, a capsule and/or coating thatresists degradation in the stomach. In other embodiments, thepharmaceutical composition comprises one of the solid formulationsdescribed herein. In certain embodiments, the pharmaceutical compositionis administered to the subject for an extended time.

In yet another aspect, a use of a combination of ingredients describedherein is provided in the preparation of a medicament for reversing, orin another aspect reducing the incidence of, endothelial dysfunction.

Still another aspect provides a method of reversing, or in anotheraspect reducing the incidence of, endothelial dysfunction, the methodcomprising the step of administering to a subject a pharmaceuticalcomposition described hereinabove, thereby reversing or reducing theincidence of endothelial dysfunction.

Provided herein, in another embodiment, is a pharmaceutical compositionfor reversing, or in another aspect reducing the incidence of, acondition selected from a prothrombotic state and a state of plateletactivation, said pharmaceutical composition comprising insulin, a GLP-1analogue, at least one protease inhibitor, and a chelator of divalentcations. In another embodiment, the pharmaceutical composition comprisesa GLP-1 analogue, at least one protease inhibitor, and a chelator ofdivalent cations. In yet another embodiment, the pharmaceuticalcomposition comprises insulin, at least one protease inhibitor, and achelator of divalent cations. In certain embodiments, the pharmaceuticalcomposition comprises one of the oil-based liquid formulations describedherein, wherein the pharmaceutical composition may further comprise, insome embodiments, a capsule and/or coating that resists degradation inthe stomach. In other embodiments, the pharmaceutical compositioncomprises one of the solid formulations described herein. In certainembodiments, the pharmaceutical composition is administered to thesubject for an extended time.

In yet another aspect, a use of a combination of ingredients describedherein is provided in the preparation of a medicament for reversing, orin another aspect reducing the incidence of, a condition selected from aprothrombotic state and a state of platelet activation.

Still another aspect provides a method of reversing, or in anotheraspect reducing the incidence of, a condition selected from aprothrombotic state and a state of platelet activation, the methodcomprising the step of administering to a subject a pharmaceuticalcomposition described hereinabove, thereby reversing or reducing theincidence of a condition selected from a prothrombotic state and a stateof platelet activation.

Provided herein, in another embodiment, is a pharmaceutical compositionfor reversing, or in another aspect reducing the incidence of,sub-clinical systemic inflammation, said pharmaceutical compositioncomprising insulin, a GLP-1 analogue, at least one protease inhibitor,and a chelator of divalent cations. In another embodiment, thepharmaceutical composition comprises a GLP-1 analogue, at least oneprotease inhibitor, and a chelator of divalent cations. In yet anotherembodiment, the pharmaceutical composition comprises insulin, at leastone protease inhibitor, and a chelator of divalent cations. In certainembodiments, the pharmaceutical composition comprises one of theoil-based liquid formulations described herein, wherein thepharmaceutical composition may further comprise, in some embodiments, acapsule and/or coating that resists degradation in the stomach. In otherembodiments, the pharmaceutical composition comprises one of the solidformulations described herein. In certain embodiments, thepharmaceutical composition is administered to the subject for anextended time.

In yet another aspect, a use of a combination of ingredients describedherein is provided in the preparation of a medicament for reversing, orin another aspect reducing the incidence of, sub-clinical systemicinflammation.

Still another aspect provides a method of reversing, or in anotheraspect reducing the incidence of, sub-clinical systemic inflammation,the method comprising the step of administering to a subject apharmaceutical composition described hereinabove, thereby reversing orreducing the incidence of sub-clinical systemic inflammation.

Provided herein, in another embodiment, is a pharmaceutical compositionfor reducing the incidence of, or in another aspect reversing,atherosclerosis, said pharmaceutical composition comprising insulin, aGLP-1 analogue, at least one protease inhibitor, and a chelator ofdivalent cations. In another embodiment, the pharmaceutical compositioncomprises a GLP-1 analogue, at least one protease inhibitor, and achelator of divalent cations. In yet another embodiment, thepharmaceutical composition comprises insulin, at least one proteaseinhibitor, and a chelator of divalent cations. In certain embodiments,the pharmaceutical composition comprises one of the oil-based liquidformulations described herein, wherein the pharmaceutical compositionmay further comprise, in some embodiments, a capsule and/or coating thatresists degradation in the stomach. In other embodiments, thepharmaceutical composition comprises one of the solid formulationsdescribed herein. In certain embodiments, the pharmaceutical compositionis administered to the subject for an extended time.

In yet another aspect, a use of a combination of ingredients describedherein is provided in the preparation of a medicament for reducing theincidence of, or in another aspect reversing, atherosclerosis.

Still another aspect provides a method of reducing the incidence of, orin another aspect reversing, atherosclerosis, the method comprising thestep of administering to a subject a pharmaceutical compositiondescribed hereinabove, thereby reducing the incidence of or reversingatherosclerosis.

Provided herein, in another embodiment, is a pharmaceutical compositionfor reducing the incidence of, or in another aspect preventing, obesity,which is in one embodiment central obesity, said pharmaceuticalcomposition comprising said pharmaceutical composition comprisinginsulin, a GLP-1 analogue, at least one protease inhibitor, and achelator of divalent cations. In another embodiment, the pharmaceuticalcomposition comprises a GLP-1 analogue, at least one protease inhibitor,and a chelator of divalent cations. In yet another embodiment, thepharmaceutical composition comprises insulin, at least one proteaseinhibitor, and a chelator of divalent cations. In certain embodiments,the pharmaceutical composition comprises one of the oil-based liquidformulations described herein, wherein the pharmaceutical compositionmay further comprise, in some embodiments, a capsule and/or coating thatresists degradation in the stomach. In other embodiments, thepharmaceutical composition comprises one of the solid formulationsdescribed herein. In certain embodiments, the pharmaceutical compositionis administered to the subject for an extended time.

In yet another aspect, a use of a combination of ingredients describedherein is provided in the preparation of a medicament for reducing theincidence of, or in another aspect preventing, obesity.

Still another aspect provides a method of reducing obesity in a subjectwith hepatic steatosis, the method comprising the step of administeringto a subject with hepatic steatosis a pharmaceutical compositiondescribed hereinabove, thereby reducing obesity.

Provided herein, in another embodiment, is a pharmaceutical compositionfor reducing hepatic insulin resistance in a subject with hepaticsteatosis, said pharmaceutical composition comprising insulin, a GLP-1analogue, at least one protease inhibitor, and a chelator of divalentcations. In another embodiment, the pharmaceutical composition comprisesa GLP-1 analogue, at least one protease inhibitor, and a chelator ofdivalent cations. In yet another embodiment, the pharmaceuticalcomposition comprises insulin, at least one protease inhibitor, and achelator of divalent cations. In certain embodiments, the pharmaceuticalcomposition comprises one of the oil-based liquid formulations describedherein, wherein the pharmaceutical composition may further comprise, insome embodiments, a capsule and/or coating that resists degradation inthe stomach. In other embodiments, the pharmaceutical compositioncomprises one of the solid formulations described herein. In certainembodiments, the pharmaceutical composition is administered to thesubject for an extended time.

In yet another aspect, a use of a combination of ingredients describedherein is provided in the preparation of a medicament for reducinghepatic insulin resistance in a subject with hepatic steatosis.

Still another aspect provides a method of reducing hepatic insulinresistance in a subject with hepatic steatosis, the method comprisingthe step of administering to a subject with hepatic steatosis apharmaceutical composition described hereinabove, thereby reducinghepatic insulin resistance in a subject with hepatic steatosis.

The oil, insulin, GLP-1 analogue, protease inhibitor(s), chelator,coating, and other optional ingredients of the described methods andcompositions may be any of those described herein; each alternative maybe combined freely to form discrete embodiments of the inventiondisclosed herein.

“Liquid” as used herein refers to a phase that flows freely and has aconstant volume under ambient conditions. Fish oil, for instance, is aliquid under ambient conditions. The term includes oil-based solutions,suspensions, and combinations thereof. In alternative embodiments, theterm may refers to a composition that has a viscosity within the rangeof 1-1000 millipascal seconds, inclusive, at 20° C.

In embodiments where both a GLP-1 analogue and insulin are both present,these two components are indicated for co-administration together,either in the same or in separate dosage forms. In the case of separatedosage forms, “co-administration” in this regard, may refer either tosimultaneous administration or, in another embodiment, to administrationwithin 30 minutes of each other. In still other embodiments, thedifferent components are indicated for administration in a particularorder, separated by a set time interval that will typically be 30minutes or less. For example, the insulin-containing dosage form may beindicated for administration 2-10 minutes after the exenatide-containingdosage form; in other embodiments, 10-20 minutes after theexenatide-containing dosage form; in other embodiments, 20-30 minutesafter the exenatide-containing dosage form; and in other embodiments,30-60 minutes after the exenatide-containing dosage form. Oral dosageforms such as those provided herein lend themselves to sequentialadministration more than injected dosage forms, since regimens requiringrepeated injections are likely to be associated with low rates ofcompliance.

Reference herein to “extended” administration may refer, in variousembodiments, to administration for more than 1 month, more than 6 weeks,more than 2 months, more than 3 months, more than 4 months, more than 5months, more than 6 months, more than 7 months, more than 8 months, morethan 9 months, more than 10 months, more than 12 months, more than 15months, more than 18 months, more than 24 months, more than 30 months,or more than 36 months, more than 48 months, more than 60 months, morethan 72 months, more than 96 months, more than 10 years, more than 15years, or more than 20 years. In other embodiments, the term may referto administration for 1-60 months, 2-60 months, 3-60 months, 4-60months, 5-60 months, 6-60 months, 8-60 months, 10-60 months, 12-60months, 1-36 months, 2-36 months, 3-36 months, 4-36 months, 5-36 months,6-36 months, 8-36 months, 10-36 months, 12-36 months, 1-36 months, 2-24months, 3-24 months, 4-24 months, 5-24 months, 6-24 months, 8-24 months,10-24 months, 12-24 months, 1-120 months, 2-120 months, 3-120 months,4-120 months, 5-120 months, 6-120 months, 8-120 months, 10-120 months,12-120 months, 1-240 months, 2-240 months, 3-240 months, 4-240 months,5-240 months, 6-240 months, 8-240 months, 10-240 months, or 12-240months.

In more specific embodiments, between 8-16 mg of insulin is administeredonce per day for more than 1 month, more than 6 weeks, more than 2months, more than 3 months, more than 4 months, more than 5 months, morethan 6 months, more than 7 months, more than 8 months, more than 9months, more than 10 months, more than 12 months, more than 15 months,more than 18 months, more than 24 months, more than 30 months, or morethan 36 months, more than 48 months, more than 60 months, more than 72months, more than 96 months, more than 10 years, more than 15 years, ormore than 20 years. In other embodiments, the term may refer toadministration for 1-60 months, 2-60 months, 3-60 months, 4-60 months,5-60 months, 6-60 months, 8-60 months, 10-60 months, 12-60 months, 1-36months, 2-36 months, 3-36 months, 4-36 months, 5-36 months, 6-36 months,8-36 months, 10-36 months, 12-36 months, 1-36 months, 2-24 months, 3-24months, 4-24 months, 5-24 months, 6-24 months, 8-24 months, 10-24months, 12-24 months, 1-120 months, 2-120 months, 3-120 months, 4-120months, 5-120 months, 6-120 months, 8-120 months, 10-120 months, 12-120months, 1-240 months, 2-240 months, 3-240 months, 4-240 months, 5-240months, 6-240 months, 8-240 months, 10-240 months, or 12-240 months.

In still other embodiments, between 8-16 mg of insulin is administeredtwice per day for more than 1 month, more than 6 weeks, more than 2months, more than 3 months, more than 4 months, more than 5 months, morethan 6 months, more than 7 months, more than 8 months, more than 9months, more than 10 months, more than 12 months, more than 15 months,more than 18 months, more than 24 months, more than 30 months, or morethan 36 months, more than 48 months, more than 60 months, more than 72months, more than 96 months, more than 10 years, more than 15 years, ormore than 20 years. In other embodiments, the term may refer toadministration for 1-60 months, 2-60 months, 3-60 months, 4-60 months,5-60 months, 6-60 months, 8-60 months, 10-60 months, 12-60 months, 1-36months, 2-36 months, 3-36 months, 4-36 months, 5-36 months, 6-36 months,8-36 months, 10-36 months, 12-36 months, 1-36 months, 2-24 months, 3-24months, 4-24 months, 5-24 months, 6-24 months, 8-24 months, 10-24months, 12-24 months, 1-120 months, 2-120 months, 3-120 months, 4-120months, 5-120 months, 6-120 months, 8-120 months, 10-120 months, 12-120months, 1-240 months, 2-240 months, 3-240 months, 4-240 months, 5-240months, 6-240 months, 8-240 months, 10-240 months, or 12-240 months.

In yet other embodiments, between 300-600 mcg of exenatide isadministered once per day, for more than 1 month, more than 6 weeks,more than 2 months, more than 3 months, more than 4 months, more than 5months, more than 6 months, more than 7 months, more than 8 months, morethan 9 months, more than 10 months, more than 12 months, more than 15months, more than 18 months, more than 24 months, more than 30 months,or more than 36 months, more than 48 months, more than 60 months, morethan 72 months, more than 96 months, more than 10 years, more than 15years, or more than 20 years. In other embodiments, the term may referto administration for 1-60 months, 2-60 months, 3-60 months, 4-60months, 5-60 months, 6-60 months, 8-60 months, 10-60 months, 12-60months, 1-36 months, 2-36 months, 3-36 months, 4-36 months, 5-36 months,6-36 months, 8-36 months, 10-36 months, 12-36 months, 1-36 months, 2-24months, 3-24 months, 4-24 months, 5-24 months, 6-24 months, 8-24 months,10-24 months, 12-24 months, 1-120 months, 2-120 months, 3-120 months,4-120 months, 5-120 months, 6-120 months, 8-120 months, 10-120 months,12-120 months, 1-240 months, 2-240 months, 3-240 months, 4-240 months,5-240 months, 6-240 months, 8-240 months, 10-240 months, or 12-240months.

In yet other embodiments, between 300-600 mcg of exenatide isadministered twice per day, for more than 1 month, more than 6 weeks,more than 2 months, more than 3 months, more than 4 months, more than 5months, more than 6 months, more than 7 months, more than 8 months, morethan 9 months, more than 10 months, more than 12 months, more than 15months, more than 18 months, more than 24 months, more than 30 months,or more than 36 months, more than 48 months, more than 60 months, morethan 72 months, more than 96 months, more than 10 years, more than 15years, or more than 20 years. In other embodiments, the term may referto administration for 1-60 months, 2-60 months, 3-60 months, 4-60months, 5-60 months, 6-60 months, 8-60 months, 10-60 months, 12-60months, 1-36 months, 2-36 months, 3-36 months, 4-36 months, 5-36 months,6-36 months, 8-36 months, 10-36 months, 12-36 months, 1-36 months, 2-24months, 3-24 months, 4-24 months, 5-24 months, 6-24 months, 8-24 months,10-24 months, 12-24 months, 1-120 months, 2-120 months, 3-120 months,4-120 months, 5-120 months, 6-120 months, 8-120 months, 10-120 months,12-120 months, 1-240 months, 2-240 months, 3-240 months, 4-240 months,5-240 months, 6-240 months, 8-240 months, 10-240 months, or 12-240months.

In yet other embodiments, between 300-600 mcg of exenatide isadministered three times per day, or in other embodiments up to 3 timesper day, for more than 1 month, more than 6 weeks, more than 2 months,more than 3 months, more than 4 months, more than 5 months, more than 6months, more than 7 months, more than 8 months, more than 9 months, morethan 10 months, more than 12 months, more than 15 months, more than 18months, more than 24 months, more than 30 months, or more than 36months, more than 48 months, more than 60 months, more than 72 months,more than 96 months, more than 10 years, more than 15 years, or morethan 20 years. In other embodiments, the term may refer toadministration for 1-60 months, 2-60 months, 3-60 months, 4-60 months,5-60 months, 6-60 months, 8-60 months, 10-60 months, 12-60 months, 1-36months, 2-36 months, 3-36 months, 4-36 months, 5-36 months, 6-36 months,8-36 months, 10-36 months, 12-36 months, 1-36 months, 2-24 months, 3-24months, 4-24 months, 5-24 months, 6-24 months, 8-24 months, 10-24months, 12-24 months, 1-120 months, 2-120 months, 3-120 months, 4-120months, 5-120 months, 6-120 months, 8-120 months, 10-120 months, 12-120months, 1-240 months, 2-240 months, 3-240 months, 4-240 months, 5-240months, 6-240 months, 8-240 months, 10-240 months, or 12-240 months.

Once-per-day administration, as used herein, can refer to administrationduring any time of day, or in other embodiments, administration each dayat a specific time of day. In certain embodiments, once per dayadministration may be before bedtime.

Insulin Proteins and GLP-1 Analogues

Insulin proteins and GLP-1 analogues for use as described herein are insome embodiments isolated prior to their introduction into the describedpharmaceutical compositions. “Isolated” in this regard excludesprovision of the insulin and/or GLP-1 analogue as a homogenized tissuepreparation or other form containing substantial amounts ofcontaminating proteins. An example of an isolated protein or peptide isa recombinant protein or peptide. An alternative embodiment is asynthetic protein or peptide.

A person skilled in the art will appreciate in light of the presentdisclosure that various types of insulin are suitable for the describedmethods and compositions. Exemplary insulin proteins include but are notlimited to both wild-type and mutated insulin proteins, includingsynthetic human insulin, synthetic bovine insulin, synthetic porcineinsulin, synthetic whale insulin, and metal complexes of insulin, suchas zinc complexes of insulin, protamine zinc insulin, and globin zinc.

Various classes of insulin may also be utilized, for example fast-actinginsulin, lente insulin, semilente insulin, ultralente insulin, NPHinsulin, glargine insulin, lispro insulin, aspart insulin, orcombinations of two or more of the above types of insulin.

In certain embodiments, the insulin of the described methods andcompositions is wild-type human insulin (Uniprot ID P01308). In someembodiments, human insulin is produced as a recombinant protein inbacterial cells. In other embodiments, human insulin is producedsynthetically.

GLP-1 analogues are also referred to in the art as GLP-1 mimetics. Aperson of skill in the art will appreciate that the describedcompositions may include at least one of the following GLP-1 analogues:exenatide (Byetta™; CAS no. 141732-76-5; SEQ ID NO: 4), lixisenatide(CAS no. 320367-13-3), liraglutide (CAS no. 204656-20-2), exendin-9 (CASno. 133514-43-9), AC3174 ([Leu(14)]exendin-4, Amylin Pharmaceuticals,Inc.), taspoglutide (CAS no. 275371-94-3), albiglutide (CAS no.782500-75-8), semaglutide (CAS no. 910463-68-2), LY2189265(dulaglutide™; CAS no. 923950-08-7), and CJC-1134-PC (a modifiedExendin-4 analogue conjugated to recombinant human albumin manufacturedby ConjuChem™). All CAS records were accessed on Dec. 19, 2011. Thus, incertain embodiments, the described method or composition utilizes any ofthe above-listed GLP-1 analogues. In other embodiments, one of theabove-listed GLP-1 analogues is selected. Those of skill in the art willappreciate in light of the findings of described herein that other GLP-1analogues can also be utilized.

Therapeutic insulin and GLP-1 proteins suitable for use in the presentinvention include derivatives that are modified (i.e., by the covalentattachment of a non-amino acid residue to the protein). For example, butnot by way of limitation, the protein includes proteins that have beenmodified, e.g., by glycosylation, acetylation, PEGylation,phosphorylation, amidation, or derivatization by knownprotecting/blocking groups. High-MW PEG can be attached to therapeuticproteins with or without a multifunctional linker either throughsite-specific conjugation of the PEG to the N- or C-terminus thereof orvia epsilon-amino groups present on lysine residues. Additionally, thederivative may contain one or more non-classical amino acids, forexample D-isomers of the common amino acids, 2,4-diaminobutyric acid,α-amino isobutyric acid, A-aminobutyric acid, Abu, 2-amino butyric acid,γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, Ca-methyl amino acids, and Nα-methyl amino acids.

Emulsifiers

In certain embodiments, an oil-based liquid formulation utilized in thedescribed methods and pharmaceutical compositions further comprises anemulsifier. Those skilled in the art will recognize, in light of thepresent disclosure, that a variety of pharmaceutically compatibleemulsifiers can be utilized.

In certain embodiments, the emulsifier is a component provided as amixture of (a) a monoacylglycerol (monoglyceride), a diacylglycerol(diglyceride), a triacylglycerol (triglyceride), or a mixture thereof;and (b) a polyethylene glycol (PEG) ester of a fatty acid. In thisregard, each of the terms “monoacylglycerol”, “diacylglycerol”, and“triacylglycerol” need not refer to a single compound, but rather caninclude mixtures of compounds, for example mixtures ofmonoacylglycerols, diacylglycerols, or triacylglycerols having fattyacids of varying lengths. In certain preferred embodiments,monoacylglycerols, diacylglycerols, or triacylglycerols utilized in thedescribed methods and compositions, for example those used to generalPEG esters, are from an oil source that is Generally Recognized As Safe(GRAS). Examples of GRAS oil sources are coconut oil, corn oil, peanutoil, soybean oil, Myvacet 9-45 (Diacetylated monoglycerides of C-18fatty acids).

More specific lengths of PEG moieties for use in some embodiments of thedescribed compositions and methods contain between 5-100 monomers. Inmore specific embodiments, the PEG may contain between 15-50 monomers.In still more specific embodiments, the PEG may contain between 25-40monomers. In more specific embodiments, the PEG may contain 32 monomers.

Examples of components meeting the above specifications are Gelucire™44/14, Gelucire™ 53/10, and Gelucire™ 50/13. A more specific example isGelucire™ 44/14. The suffixes 44 and 14 refer respectively to itsmelting point and its hydrophilic/lypophilic balance (HLB). Gelucire™44/14 (Gattefosse SAS, Saint-Priest, France) is obtained bypolyglycolysis of hydrogenated coconut oil (medium and long chaintriacylglycerols with PEG-32. It has a hydrophile/lipophile balance of14. It is composed of a defined admixture of C₈-C₁₈ mono-, di- andtriacylglycerols (20% w/w); PEG-32 mono- and diesters and free PEG-32(80% w/w). The main fatty acid present is lauric acid, accounting for45% on average of the total fatty acid content. It is a solid dispersioncomposed of a PEG ester fraction under a lamellar phase of 120 Å with ahelical conformation and an acylglycerol fraction under a hexagonalpacking. The main products of simulated gastrointestinal lipolysis ofGelucire™ 44/14 are PEG-32 mono and diesters.

In some embodiments, the HLB of a self-emulsifying component utilized inthe described methods and compositions is 10 or greater. In otherembodiments, it is between 11-19. In other embodiments, it is between12-18. In other embodiments, it is between 12-17. In other embodiments,it is between 12-16 inclusive, which is indicative of an oil-in-water(O/W) emulsifier. In other embodiments, it is between 13-15. In otherembodiments, it is 14. Still more specific embodiments ofself-emulsifying components have an HLB of 12-16 inclusive and comprisemedium- and long-chain triacylglycerols conjugated to PEG, freetriacylglycerols, and free PEG. In other embodiments, theself-emulsifying component has an HLB of 12-16 inclusive and consists ofa mixture of medium- and long-chain triacylglycerols conjugated to PEG,free triacylglycerols, and free PEG. In other embodiments, theself-emulsifying component has an HLB of 14 and comprises medium- andlong-chain triacylglycerols conjugated to PEG, free triacylglycerols,and free PEG. In other embodiments, the self-emulsifying component hasan HLB of 14 and consists of a mixture of medium- and long-chaintriacylglycerols conjugated to PEG, free triacylglycerols, and free PEG.

In certain embodiments, the aforementioned emulsifier, which is incertain embodiments a self-emulsifying component, constitutes 8-16%weight/weight inclusive of the oil-based liquid formulation. In morespecific embodiments, the amount is 9-15% inclusive. In more specificembodiments, the amount is 10-14% inclusive. In more specificembodiments, the amount is 11-13% inclusive. In more specificembodiments, the amount is 12%.

Non-Ionic Detergents

In certain embodiments, the oil-based liquid formulation utilized in thedescribed methods and pharmaceutical compositions further comprises apolysorbate-based detergent. Examples of polysorbate-based detergent aredetergents derived by covalently bonding polyethoxylated sorbitan to afatty acid. More specific embodiments of polysorbate-based detergentsare polysorbate-20, polysorbate-40, and polysorbate-80.

For example, polysorbate 80 (Tween-80) is a mild, non-ionic detergentderived from polyethoxylated sorbitan and oleic acid and having thefollowing structure:

In the case of polysorbate 80, the moiety shown on the right side is amixture of fatty acids, containing 60-70% oleic acid (as depicted), withthe balance being primarily linoleic, palmitic, and stearic acids.

In a more specific embodiment, the polysorbate 80 constitutes 3-10%weight/weight inclusive of an oil-based liquid formulation used in thedescribed methods and compositions. In a more specific embodiment, thepercentage is 4-8% inclusive. In a more specific embodiment, thepercentage is 4.5-6% inclusive. In a more specific embodiment, thepercentage is 5%.

Dosages

In more specific embodiments, the amount of insulin in a dosage form ofthe described methods and compositions is between 6-64 mg. In otherembodiments, the amount is between 6-56 mg. In other embodiments, theamount is between 6-48 mg. In other embodiments, the amount is between6-40 mg. In other embodiments, the amount is between 6-36 mg. In otherembodiments, the amount is between 6-32 mg. In other embodiments, theamount is between 6-28 mg. In other embodiments, the amount is between6-24 mg. In other embodiments, the amount is between 6-20 mg. In otherembodiments, the amount is between 6-14 mg. In other embodiments, theamount is between 6-12 mg. In other embodiments, the amount is between6-10 mg. In other embodiments, the amount is between 10-64 mg. In otherembodiments, the amount is between 10-56 mg. In other embodiments, theamount is between 10-48 mg. In other embodiments, the amount is between10-40 mg. In other embodiments, the amount is between 10-36 mg. In otherembodiments, the amount is between 10-32 mg. In other embodiments, theamount is between 10-28 mg. In other embodiments, the amount is between16-64 mg. In other embodiments, the amount is between 16-56 mg. In otherembodiments, the amount is between 16-48 mg. In other embodiments, theamount is between 16-40 mg. In other embodiments, the amount is between16-36 mg. In other embodiments, the amount is between 16-32 mg. In otherembodiments, the amount is between 16-28 mg. In certain embodiments, theabove dosage amounts are a daily dose.

In other embodiments, the amount is 8 mg. In other embodiments, theamount is 12 mg. In other embodiments, the amount is 16 mg. In otherembodiments, the amount is 20 mg. In other embodiments, the amount is 24mg. In other embodiments, the amount is 32 mg. In other embodiments, theamount is 40 mg. In other embodiments, the amount is 40 mg. In otherembodiments, the amount is 56 mg. In other embodiments, the amount is 64mg. In other embodiments, the amount is between 8-16 mg. In otherembodiments, the amount is between 8-14 mg. In other embodiments, theamount is between 8-12 mg. In other embodiments, the amount is between8-10 mg. In other embodiments, the amount is 16 mg. In otherembodiments, the amount is between 10-16 mg. In other embodiments, theamount is between 10-14 mg. In other embodiments, the amount is between10-18 mg. In certain embodiments, the above dosage amounts are a dailydose.

In other embodiments, the amount of insulin in a dosage form of thedescribed methods and compositions is between 0.06-0.64 mg/kg(milligrams per kilogram body weight). In other embodiments, the amountis between 0.06-0.56 mg/kg. In other embodiments, the amount is between0.06-0.48 mg/kg. In other embodiments, the amount is between 0.06-0.40mg/kg. In other embodiments, the amount is between 0.06-0.32 mg/kg. Inother embodiments, the amount is between 0.06-0.28 mg/kg. In otherembodiments, the amount is between 0.06-0.24 mg/kg. In otherembodiments, the amount is between 0.06-0.20 mg/kg. In otherembodiments, the amount is between 0.10-0.16 mg/kg. In otherembodiments, the amount is between 0.10-0.64 mg/kg. In otherembodiments, the amount is between 0.10-0.56 mg/kg. In otherembodiments, the amount is between 0.10-0.48 mg/kg. In otherembodiments, the amount is between 0.10-0.40 mg/kg. In otherembodiments, the amount is between 0.10-0.32 mg/kg. In otherembodiments, the amount is between 0.10-0.28 mg/kg. In otherembodiments, the amount is between 0.10-0.24 mg/kg. In otherembodiments, the amount is between 0.10-0.20 mg/kg. In otherembodiments, the amount is between 0.06-0.14 mg/kg. In otherembodiments, the amount is between 0.06-0.12 mg/kg. In otherembodiments, the amount is between 0.06-0.10 mg/kg. In otherembodiments, the amount is 0.08 mg/kg. In other embodiments, the amountis 0.12 mg/kg. In other embodiments, the amount is 0.16 mg/kg. In otherembodiments, the amount is between 0.08-0.16 mg/kg. In otherembodiments, the amount is between 0.08-0.14 mg/kg. In otherembodiments, the amount is between 0.08-0.12 mg/kg. In otherembodiments, the amount is between 0.08-0.10 mg/kg. In otherembodiments, the amount is between 0.10-0.16 mg/kg. In otherembodiments, the amount is between 0.10-0.18 mg/kg. In otherembodiments, the amount is between 0.10-0.14 mg/kg. In certainembodiments, the above dosage amounts are a daily dose.

In still other embodiments, the amount of insulin in the dosage form isan amount corresponding to one of the above amounts or ranges for anadult, adjusted per body weight for a pediatric patient. Adjustments ofthis kind mentioned herein utilize 62 kilograms as the adult weight. Inother embodiments, the insulin is present in an amount adjusted for apediatric patient, and the GLP-1 analogue is also present in an amountadjusted for a pediatric patient. In certain embodiments, the abovedosage amounts are a daily dose.

The dosages described herein for insulin may be for wild-type humaninsulin, or in another embodiment, for one of the other types of insulinknown in the art.

In other embodiments, the amount of a GLP-1 analogue in a dosage form ofthe described methods and compositions is 150 micrograms (mcg), 200 mcg,250 mcg, 300 mcg, 350 mcg, 400 mcg, 500 mcg, or 600 mcg. In otherembodiments, the amount of GLP-1 analogue is between 100-1600 mcginclusive for an adult patent. In other embodiments, the amount isbetween 100-1400 mcg. In other embodiments, the amount is between100-1200 mcg. In other embodiments, the amount is between 100-1000 mcg.In other embodiments, the amount is between 100-800 mcg. In otherembodiments, the amount is between 100-700 mcg. In other embodiments,the amount is between 100-600 mcg. In other embodiments, the amount isbetween 100-500 mcg. In other embodiments, the amount is between 100-400mcg. In other embodiments, the amount is between 200-1400 mcg. In otherembodiments, the amount is between 200-1400 mcg. In other embodiments,the amount is between 200-1200 mcg. In other embodiments, the amount isbetween 200-1000 mcg. In other embodiments, the amount is between200-800 mcg. In other embodiments, the amount is between 200-700 mcg. Inother embodiments, the amount is between 200-600 mcg. In otherembodiments, the amount is between 200-500 mcg. In other embodiments,the amount is between 200-400 mcg. In other embodiments, the amount isbetween 100-300 mcg. In other embodiments, the amount is between 100-250mcg. In other embodiments, the amount is between 100-200 mcg. In otherembodiments, the amount is between 100-150 mcg. In other embodiments,the amount is 100 mcg. In other embodiments, the amount is 150 mcg. Inother embodiments, the amount is 200 mcg. In other embodiments, theamount is 250 mcg. In other embodiments, the amount is 300 mcg. In otherembodiments, the amount is between 150-400 mcg. In other embodiments,the amount is between 150-300 mcg. In other embodiments, the amount isbetween 150-250 mcg. In other embodiments, the amount is between 150-200mcg. In certain embodiments, the above dosage amounts are a daily dose.In still other embodiments, the GLP-1 analogue is exenatide, present inone of the above amounts.

In other embodiments, the amount of GLP-1 analogue in a dosage form ofthe described methods and compositions is between 0.100-1.60 mcg/kg foran adult patent. In other embodiments, the amount is between 0.100-1.40mcg/kg. In other embodiments, the amount is between 0.100-1.20 mcg/kg.In other embodiments, the amount is between 0.100-1.0 mcg/kg. In otherembodiments, the amount is between 0.100-0.800 mcg/kg. In otherembodiments, the amount is between 0.100-0.700 mcg/kg. In otherembodiments, the amount is between 0.100-0.600 mcg/kg. In otherembodiments, the amount is between 0.100-0.500 mcg/kg. In otherembodiments, the amount is between 0.100-0.400 mcg/kg. In otherembodiments, the amount is between 0.200-1.40 mcg/kg. In otherembodiments, the amount is between 0.200-1.40 mcg/kg. In otherembodiments, the amount is between 0.200-1.20 mcg/kg. In otherembodiments, the amount is between 0.200-1.0 mcg/kg. In otherembodiments, the amount is between 0.200-0.800 mcg/kg. In otherembodiments, the amount is between 0.200-0.700 mcg/kg. In otherembodiments, the amount is between 0.200-0.600 mcg/kg. In otherembodiments, the amount is between 0.200-0.500 mcg/kg. In otherembodiments, the amount is between 0.200-0.400 mcg/kg. In otherembodiments, the amount is between 0.100-0.300 mcg/kg. In otherembodiments, the amount is between 0.100-0.250 mcg/kg. In otherembodiments, the amount is between 0.100-0.200 mcg/kg. In otherembodiments, the amount is between 0.100-0.150 mcg/kg. In otherembodiments, the amount is 0.100 mcg/kg. In other embodiments, theamount is 0.150 mcg/kg. In other embodiments, the amount is 0.200mcg/kg. In other embodiments, the amount is 0.250 mcg/kg. In otherembodiments, the amount is 0.300 mcg/kg. In other embodiments, theamount is between 0.150-0.400 mcg/kg. In other embodiments, the amountis between 0.150-0.300 mcg/kg. In other embodiments, the amount isbetween 0.150-0.250 mcg/kg. In other embodiments, the amount is between0.100-0.200 mcg/kg. In certain embodiments, the above dosage amounts area daily dose. In still other embodiments, the GLP-1 analogue isexenatide, present in one of the above amounts.

In other embodiments, the amount of GLP-1 analogue in the dosage form isan amount corresponding to one of the above amounts or ranges for anadult, adjusted per body weight for a pediatric patient. In otherembodiments, the GLP-1 analogue is present in an amount adjusted for apediatric patient, and the insulin is also present in an amount adjustedfor a pediatric patient, for example an amount corresponding to 4-12 mginclusive for an adult patent, adjusted for the weight of the apediatric patient. In certain embodiments, the above dosage amounts area daily dose. In still other embodiments, the GLP-1 analogue isexenatide, present in one of the above amounts.

In various embodiments, the described dosage form contains exenatide inan amount of between 100-600 mcg, 100-500 mcg, 100-400 mcg, 100-300 mcg,200-600 mcg, 200-500 mcg, 200-400 mcg, 200-300 mcg, 150-300 mcg, or150-250 mcg; together with 8-16 mg insulin. In other embodiments, thedescribed dosage form contains exenatide in an amount of between 100-600mcg, 100-500 mcg, 100-400 mcg, 100-300 mcg, 200-600 mcg, 200-500 mcg,200-400 mcg, 200-300 mcg, 150-300 mcg, or 150-250 mcg; together with8-12 mg insulin. In other embodiments, the described dosage formcontains exenatide in an amount of between 100-600 mcg, 100-500 mcg,100-400 mcg, 100-300 mcg, 200-600 mcg, 200-500 mcg, 200-400 mcg, 200-300mcg, 150-300 mcg, or 150-250 mcg; together with 12-16 mg insulin. Inother embodiments, the described dosage form contains exenatide in anamount of between 100-600 mcg, 100-500 mcg, 100-400 mcg, 100-300 mcg,200-600 mcg, 200-500 mcg, 200-400 mcg, 200-300 mcg, 150-300 mcg, or150-250 mcg; together with 16-24 mg insulin. In other embodiments, thedescribed dosage form contains exenatide in an amount of between 100-600mcg, 100-500 mcg, 100-400 mcg, 100-300 mcg, 200-600 mcg, 200-500 mcg,200-400 mcg, 200-300 mcg, 150-300 mcg, or 150-250 mcg; together with24-32 mg insulin. In other embodiments, the described dosage formcontains exenatide in an amount of between 100-600 mcg, 100-500 mcg,100-400 mcg, 100-300 mcg, 200-600 mcg, 200-500 mcg, 200-400 mcg, 200-300mcg, 150-300 mcg, or 150-250 mcg; together with 12-16 mg insulin. Inother embodiments, the described dosage form contains exenatide in anamount of between 100-600 mcg, 100-500 mcg, 100-400 mcg, 100-300 mcg,200-600 mcg, 200-500 mcg, 200-400 mcg, 200-300 mcg, 150-300 mcg, or150-250 mcg; together with 8 mg insulin. In other embodiments, thedescribed dosage form contains exenatide in an amount of between 100-600mcg, 100-500 mcg, 100-400 mcg, 100-300 mcg, 200-600 mcg, 200-500 mcg,200-400 mcg, 200-300 mcg, 150-300 mcg, or 150-250 mcg; together with 12mg insulin. In other embodiments, the described dosage form containsexenatide in an amount of between 100-600 mcg, 100-500 mcg, 100-400 mcg,100-300 mcg, 200-600 mcg, 200-500 mcg, 200-400 mcg, 200-300 mcg, 150-300mcg, or 150-250 mcg; together with 16 mg insulin. In other embodiments,the described dosage form contains exenatide in an amount of between100-600 mcg, 100-500 mcg, 100-400 mcg, 100-300 mcg, 200-600 mcg, 200-500mcg, 200-400 mcg, 200-300 mcg, 150-300 mcg, or 150-250 mcg; togetherwith 20 mg insulin. In other embodiments, the described dosage formcontains exenatide in an amount of between 100-600 mcg, 100-500 mcg,100-400 mcg, 100-300 mcg, 200-600 mcg, 200-500 mcg, 200-400 mcg, 200-300mcg, 150-300 mcg, or 150-250 mcg; together with 24 mg insulin. In otherembodiments, the described dosage form contains exenatide in an amountof between 100-600 mcg, 100-500 mcg, 100-400 mcg, 100-300 mcg, 200-600mcg, 200-500 mcg, 200-400 mcg, 200-300 mcg, 150-300 mcg, or 150-250 mcg;together with 28 mg insulin. In other embodiments, the described dosageform contains exenatide in an amount of between 100-600 mcg, 100-500mcg, 100-400 mcg, 100-300 mcg, 200-600 mcg, 200-500 mcg, 200-400 mcg,200-300 mcg, 150-300 mcg, or 150-250 mcg; together with 32 mg insulin.In certain embodiments, the above dosage amounts are a daily dose.

In other embodiments, the described dosage form contains insulin in anamount of 8-32 mg, 8-28 mg, 8-24 mg, 8-20 mg, 8-16 mg, 8-12 mg, 12-32mg, 16-32 mg, 20-32 mg, 24-32 mg, 12-24 mg, 16-24 mg, 12-20 mg, or 16-20mg; together with 150-300 mcg exenatide. In other embodiments, thedescribed dosage form contains insulin in an amount of 8-32 mg, 8-28 mg,8-24 mg, 8-20 mg, 8-16 mg, 8-12 mg, 12-32 mg, 16-32 mg, 20-32 mg, 24-32mg, 12-24 mg, 16-24 mg, 12-20 mg, or 16-20 mg; together with 300-450 mcgexenatide. In other embodiments, the described dosage form containsinsulin in an amount of 8-32 mg, 8-28 mg, 8-24 mg, 8-20 mg, 8-16 mg,8-12 mg, 12-32 mg, 16-32 mg, 20-32 mg, 24-32 mg, 12-24 mg, 16-24 mg,12-20 mg, or 16-20 mg; together with 450-600 mcg exenatide. In otherembodiments, the described dosage form contains insulin in an amount of8-32 mg, 8-28 mg, 8-24 mg, 8-20 mg, 8-16 mg, 8-12 mg, 12-32 mg, 16-32mg, 20-32 mg, 24-32 mg, 12-24 mg, 16-24 mg, 12-20 mg, or 16-20 mg;together with 100-150 mcg exenatide. In other embodiments, the describeddosage form contains insulin in an amount of 8-32 mg, 8-28 mg, 8-24 mg,8-20 mg, 8-16 mg, 8-12 mg, 12-32 mg, 16-32 mg, 20-32 mg, 24-32 mg, 12-24mg, 16-24 mg, 12-20 mg, or 16-20 mg; together with 150-200 mcgexenatide. In other embodiments, the described dosage form containsinsulin in an amount of 8-32 mg, 8-28 mg, 8-24 mg, 8-20 mg, 8-16 mg,8-12 mg, 12-32 mg, 16-32 mg, 20-32 mg, 24-32 mg, 12-24 mg, 16-24 mg,12-20 mg, or 16-20 mg; together with 200-250 mcg exenatide. In otherembodiments, the described dosage form contains insulin in an amount of8-32 mg, 8-28 mg, 8-24 mg, 8-20 mg, 8-16 mg, 8-12 mg, 12-32 mg, 16-32mg, 20-32 mg, 24-32 mg, 12-24 mg, 16-24 mg, 12-20 mg, or 16-20 mg;together with 250-300 mcg exenatide. In certain embodiments, the abovedosage amounts are a daily dose.

In other embodiments, the described dosage form contains insulin in anamount of 8-32 mg, 8-28 mg, 8-24 mg, 8-20 mg, 8-16 mg, 8-12 mg, 12-32mg, 16-32 mg, 20-32 mg, 24-32 mg, 12-24 mg, 16-24 mg, 12-20 mg, or 16-20mg; together with 100 mcg exenatide. In other embodiments, the describeddosage form contains insulin in an amount of 8-32 mg, 8-28 mg, 8-24 mg,8-20 mg, 8-16 mg, 8-12 mg, 12-32 mg, 16-32 mg, 20-32 mg, 24-32 mg, 12-24mg, 16-24 mg, 12-20 mg, or 16-20 mg; together with 200 mcg exenatide. Inother embodiments, the described dosage form contains insulin in anamount of 8-32 mg, 8-28 mg, 8-24 mg, 8-20 mg, 8-16 mg, 8-12 mg, 12-32mg, 16-32 mg, 20-32 mg, 24-32 mg, 12-24 mg, 16-24 mg, 12-20 mg, or 16-20mg; together with 250 mcg exenatide. In other embodiments, the describeddosage form contains insulin in an amount of 8-32 mg, 8-28 mg, 8-24 mg,8-20 mg, 8-16 mg, 8-12 mg, 12-32 mg, 16-32 mg, 20-32 mg, 24-32 mg, 12-24mg, 16-24 mg, 12-20 mg, or 16-20 mg; together with 300 mcg exenatide. Inother embodiments, the described dosage form contains insulin in anamount of 8-32 mg, 8-28 mg, 8-24 mg, 8-20 mg, 8-16 mg, 8-12 mg, 12-32mg, 16-32 mg, 20-32 mg, 24-32 mg, 12-24 mg, 16-24 mg, 12-20 mg, or 16-20mg; together with 400 mcg exenatide. In other embodiments, the describeddosage form contains insulin in an amount of 8-32 mg, 8-28 mg, 8-24 mg,8-20 mg, 8-16 mg, 8-12 mg, 12-32 mg, 16-32 mg, 20-32 mg, 24-32 mg, 12-24mg, 16-24 mg, 12-20 mg, or 16-20 mg; together with 500 mcg exenatide. Inother embodiments, the described dosage form contains insulin in anamount of 8-32 mg, 8-28 mg, 8-24 mg, 8-20 mg, 8-16 mg, 8-12 mg, 12-32mg, 16-32 mg, 20-32 mg, 24-32 mg, 12-24 mg, 16-24 mg, 12-20 mg, or 16-20mg; together with 600 mcg exenatide. In certain embodiments, the abovedosage amounts are a daily dose.

In other embodiments, the described dosage form contains 8-16 mg insulinand 150-300 mcg. exenatide. In other embodiments, the described dosageform contains 8-12 mg insulin and 150-300 mcg. exenatide. In otherembodiments, the described dosage form contains 12-16 mg insulin and150-300 mcg. exenatide. In other embodiments, the described dosage formcontains 6-16 mg insulin and 150-300 mcg. exenatide. In certainembodiments, the above dosage amounts are a daily dose.

In other embodiments, the described dosage form contains 8-16 mg insulinand 100-400 mcg. exenatide. In other embodiments, the described dosageform contains 8-12 mg insulin and 100-400 mcg. exenatide. In otherembodiments, the described dosage form contains 12-16 mg insulin and100-400 mcg. exenatide. In other embodiments, the described dosage formcontains 6-16 mg insulin and 100-400 mcg. exenatide. In certainembodiments, the above dosage amounts are a daily dose.

In other embodiments, the described dosage form contains 8-16 mg insulinand 100-200 mcg. exenatide. In other embodiments, the described dosageform contains 8-12 mg insulin and 100-200 mcg. exenatide. In otherembodiments, the described dosage form contains 12-16 mg insulin and100-200 mcg. exenatide. In other embodiments, the described dosage formcontains 6-16 mg insulin and 100-200 mcg. exenatide. In certainembodiments, the above dosage amounts are a daily dose.

In other embodiments, the described dosage form contains 8-16 mg insulinand 200-400 mcg. exenatide. In other embodiments, the described dosageform contains 8-12 mg insulin and 200-400 mcg. exenatide. In otherembodiments, the described dosage form contains 12-16 mg insulin and200-400 mcg. exenatide. In other embodiments, the described dosage formcontains 6-16 mg insulin and 200-400 mcg. exenatide. In certainembodiments, the above dosage amounts are a daily dose.

In other embodiments, the described dosage form contains 8-16 mg insulinand 150-300 mcg. exenatide. In certain embodiments, the above dosageamounts are a daily dose.

Protease Inhibitors

Those skilled in the art will appreciate, in light of the presentdisclosure, that a variety or protease inhibitors may be used to protectthe GLP-1 analogue and/or insulin in the described formulations. Incertain embodiments, the protease inhibitor present in the describedcompositions is selected from a trypsin inhibitor and a chymotrypsininhibitor. In more particular embodiments, the inhibitor is a trypsininhibitor or, in other embodiments, is a chymotrypsin inhibitor. Instill other embodiments, the pharmaceutical composition contains one ormore inhibitors that collectively inhibit both trypsin and chymotrypsin.In more particular embodiments, both a trypsin inhibitor and achymotrypsin inhibitor are present; or, in other embodiments, theprotease inhibitor is a single inhibitor that inhibits both trypsin andchymotrypsin. An example of a single inhibitor that inhibits bothtrypsin and chymotrypsin, not intended to be limiting, is BBI (describedherein).

As used herein, the term “chymotrypsin inhibitor” refers to any agentcapable of inhibiting the action of chymotrypsin on a substrate. Theability of an agent to inhibit chymotrypsin can be measured using assayswell known in the art. For example, in a typical assay, one unitcorresponds to the amount of inhibitor that reduces the trypsin activityby one benzoyl-L-arginine ethyl ester unit (BAEE-U). One BAEE-U is theamount of enzyme that increases the absorbance at 253 nm by 0.001 perminute at pH 7.6 and 25° C. See, for example, K. Ozawa, M. Laskowski,1966, J. Biol. Chem. 241:3955; and Y. Birk, 1976, Meth. Enzymol. 45:700.

Unless indicated otherwise, anti-chymotrypsin activity referred toherein is measured using chymotrypsin having an activity of 40 BTEEunits per mg. of chymotrypsin, and is expressed in mg. of chymotrypsininhibited per mg. of protein being tested. BTEE refers toN-Benzoyl-L-Tyrosine Ethyl Ester (see the directions for Sigma-AldrichProduct No. B6125).

As used herein, the term “trypsin inhibitor” refers to any agent capableof inhibiting the action of trypsin on a substrate. The ability of anagent to inhibit trypsin can be measured using assays well known in theart. For example, in a typical assay, one unit corresponds to the amountof inhibitor that reduces the trypsin activity by one benzoyl-L-arginineethyl ester unit (BAEE-U). One BAEE-U is the amount of enzyme thatincreases the absorbance at 253 nm by 0.001 per minute at pH 7.6 and 25°C. See, for example, K. Ozawa, M. Laskowski, 1966, J. Biol. Chem.241:3955; and Y. Birk, 1976, Meth. Enzymol. 45:700. An example of aninhibitor that inhibits trypsin, not intended to be limiting, is KTI3(described herein).

Unless indicated otherwise, anti-trypsin activity referred to herein ismeasured using trypsin having an activity of 10,000 BAEE units per mg.of trypsin, and is expressed in mg. of trypsin inhibited per mg. ofprotein being tested. BAEE refers to Na-Benzoyl-L-Arginine Ethyl EsterSolution (see the directions for Sigma-Aldrich Product No. B4500). Forexample, in a typical assay, one unit corresponds to the amount ofinhibitor that reduces the trypsin activity by one benzoyl-L-arginineethyl ester unit (BAEE-U). One BAEE-U is the amount of enzyme thatincreases the absorbance at 253 nm by 0.001 per minute at pH 7.6 and 25°C. See, for example, K. Ozawa, M. Laskowski, 1966, J. Biol. Chem.241:3955; and Y. Birk, 1976, Meth. Enzymol. 45:700.

Some trypsin inhibitors known in the art are specific to trypsin, whileothers inhibit trypsin and other proteases such as chymotrypsin. Trypsininhibitors can be derived from animal or vegetable sources: for example,soybean, corn, lima and other beans, squash, sunflower, bovine and otheranimal pancreas and lung, chicken and turkey egg white, soy-based infantformula, and mammalian blood. Trypsin inhibitors can also be ofmicrobial origin: for example, antipain; see, for example, H. Umezawa,1976, Meth. Enzymol. 45, 678. A trypsin inhibitor can also be anarginine or lysine mimic or other synthetic compound: for examplearylguanidine, benzamidine, 3,4-dichloroisocoumarin,diisopropylfluorophosphate, gabexate mesylate, or phenylmethanesulfonylfluoride. As used herein, an arginine or lysine mimic is a compound thatis capable of binding to the P¹ pocket of trypsin and/or interferingwith trypsin active site function.

In certain embodiments, in cases where a trypsin inhibitor is utilizedin methods and compositions of the present invention, the trypsininhibitor is selected from the group consisting of lima bean trypsininhibitor, aprotinin, (a.k.a. pancreatic trypsin inhibitor or basicpancreatic trypsin inhibitor [BPTI]; Uniprot No. P00974 [databaseaccessed on January 2, 2013]), Kazal inhibitor (pancreatic secretorytrypsin inhibitor), ovomucoid, Alpha 1-antitrypsin, Cortisol bindingglobulin, Centerin ([SERPINA9/GCET1 (germinal centre B-cell-expressedtranscript 1)], PI-6 (Sun et al 1995), PI-8 (Sprecher et al 1995),Bomapin, a Glade A serpin [for example Serpina3 (NCBI Gene ID: 12;database accessed on Dec. 27, 2012), Serpina6 (NCBI Gene ID: 866;database accessed on Dec. 27, 2012), Serpina12 (NCBI Gene ID: 145264;database accessed on Dec 27, 2012); Serpinal0 (NCBI Gene ID: 51156;database accessed on Dec. 27, 2012); Serpina7 (NCBI Gene ID: 6906;database accessed on Dec. 27, 2012); Serpina9 (NCBI Gene ID: 327657;database accessed on Dec. 27, 2012); Serpinal 1 (NCBI Gene ID: 256394;database accessed on Dec. 27, 2012); Serpina13 (NCBI Gene ID: 388007;database accessed on Dec. 27, 2012); Serpina2 (NCBI Gene ID: 390502;database accessed on Dec. 27, 2012); and Serpina4 (NCBI Gene ID: 5104;database accessed on Dec. 27, 2012)] Yukopin (SerpinB12; Gene ID: 89777;database accessed on Dec. 27, 2012), antipain, benzamidine,3,4-dichloroisocoumarin, diisopropylfluorophosphate, and gabexatemesylate. In other embodiments, more than one, for example 2, 3, or 4,of the above inhibitors is selected.

A representative precursor sequence of aprotinin is:

(SEQ ID NO: 1) MKMSRLCLSV ALLVLLGTLA ASTPGCDTSN QAKAQRPDFCLEPPYTGPCK ARIIRYFYNA KAGLCQTFVY GGCRAKRNNF KSAEDCMRTC GGAIGPWENL.

Of these 100 residues, residues 1-21 are the signal peptide, 22-35 and94-100 are propeptides, and the mature chain aprotinin chain is composedof residues 36-93 (58 AA).

In certain embodiments, in cases where a chymotrypsin inhibitor isutilized in methods and compositions of the present invention, thechymotrypsin inhibitor is selected from the group consisting of soybeantrypsin inhibitor, Bowman-Birk inhibitor, aprotinin, N-Acetyl-eglin Cfrom leeches (Sigma-Aldrich cat. no. E7888), chymostatin (Sigma-Aldrichcat. no. C7268), α1-antitrypsin (Sigma-Aldrich cat. no. A9024),α1-antichymotrypsin (Sigma-Aldrich cat. no. A9285), potato type Iproteinase inhibitor, and potato type II proteinase inhibitor.

In other embodiments, the chymotrypsin inhibitor and/or the trypsininhibitor is derived from soybean. Chymotrypsin and trypsin inhibitorsderived from soybean (Glycine max) are readily available and areconsidered to be safe for human consumption. They include SBTI (soybeantrypsin inhibitor), which in its natural form is composed of KTI3(Kunitz Trypsin Inhibitor 3), which inhibits trypsin, and BBI(Bowman-Birk inhibitor; Uniprot number P01055 [database accessed on Jan.3, 2013]), which inhibits chymotrypsin and trypsin. The term “SBTI”, asused herein, refers, unless indicated otherwise, to a combination ofKTI3 and BBI, which may be a naturally occurring combination or anartificial combination of KTI3 and BBI. In accordance with acceptedusage in the art, SBTI is sometimes referred to herein as a singleprotease inhibitor, despite the fact that it has two separate proteasecomponents.

Chymotrypsin and trypsin inhibitor are available for example fromSigma-Aldrich, St. Louis, Mo., USA.

In certain embodiments, SBTI is present in an amount between 25-125 mgper dosage unit when present with another inhibitor of chymotrypsinand/or trypsin and 75-200 mg per dosage unit when present as the onlyinhibitor of chymotrypsin and/or trypsin. In various embodiments, SBTIis present in an amount of 30-180 mg, 35-170 mg, 40-160 mg, 45-150 mg,50-140 mg, 50-130 mg, 50-120 mg, 50-100 mg, 55-95 mg, 60-90 mg, 65-85mg, 70-80 mg, 50-150 mg, 60-140 mg, 70-130 mg, 80-120 mg, 90-100 mg,100-150 mg, 110-140 mg, 120-130 mg, 100-200 mg, 110-190 mg, 120-180 mg,130-170 mg, or 140-160 mg per dosage unit. All the aforementioned rangesare inclusive. In still other embodiments, the SBTI is present in anamount of 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 75 mg, 90 mg, 100 mg, 110mg, 125 mg, 150 mg, 175 mg, or 200 mg per dosage unit.

Methods for preparing BBI are described for example in U.S. Pat. No.7,404,973 and in PCT International Application Publ. No. WO 2013/10289to Avraham Hershko, filed on Jan. 31, 2013, the contents of which areincorporated herein by reference.

A representative precursor sequence of BBI is:

(SEQ ID NO: 2) MVVLKVCLVL LFLVGGTTSA NLRLSKLGLL MKSDHQHSNDDESSKPCCDQ CACTKSNPPQ CRCSDMRLNS CHSACKSCICALSYPAQCFC VDITDFCYEP CKPSEDDKEN.

Of these 110 residues, residues 1-19 are the signal peptide, 20-39 are apropeptide, and the mature chain BBI chain is composed of residues40-110 (71 AA).

In certain embodiments, BBI is present in an amount between 25-125 mgper dosage unit when present with another inhibitor of chymotrypsinand/or trypsin and 75-200 mg per dosage unit when present as the onlyinhibitor of chymotrypsin and/or trypsin. In various embodiments, BBI ispresent in an amount of 30-180 mg, 35-170 mg, 40-160 mg, 45-150 mg,50-140 mg, 50-130 mg, 50-120 mg, 50-100 mg, 55-95 mg, 60-90 mg, 65-85mg, 70-80 mg, 50-150 mg, 60-140 mg, 70-130 mg, 80-120 mg, 90-100 mg,100-150 mg, 110-140 mg, 120-130 mg, 100-200 mg, 110-190 mg, 120-180 mg,130-170 mg, or 140-160 mg per dosage unit. All the aforementioned rangesare inclusive. In still other embodiments, the BBI is present in anamount of 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 75 mg, 90 mg, 100 mg, 110mg, 125 mg, 150 mg, 175 mg, or 200 mg per dosage unit.

In one aspect, a BBI utilized in the described methods and compositionsis at least 85% pure as measured, in various embodiments, by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), BrilliantBlue staining, or imager quantitation. In certain embodiments, thesoybean product is soy flour.

In yet another aspect, the protein content of the BBI is greater than95% by BCA (bicinchoninic acid) assay.

In yet another aspect the BBI utilized in the described methods andcompositions contains less than 0.1% high-MW contaminants, for exampleas assessed by SDS-PAGE and imager quantitation.

In other embodiments, the isolated BBI has an anti-chymotrypsin activityof at least 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, or 1.4 mg. chymotrypsininhibited per mg. inhibitor. In other embodiments, the anti-chymotrypsinactivity is in the range of 0.8-1.8, 0.9-1.8, 1.0-1.8, 1.1-1.8, 1.2-1.8,1.3-1.8, or 1.4-1.8 mg. chymotrypsin inhibited per mg. inhibitor. Inmore specific embodiments, the activity is 0.8-1.8 mg. chymotrypsininhibited per mg. inhibitor. In other embodiments, the activity is inthe range of 0.8-1.5 mg. chymotrypsin inhibited per mg. inhibitor. Inthe context of a pharmaceutical composition, this value refers tocharacteristics of the BBI prior to its being mixed with one or moreother components of the pharmaceutical composition.

Those skilled in the art will appreciate that each of the above purityrequirements, regarding its protein content, level of contaminants, orpotency, is typically assessed prior to the BBI being mixed with any ofthe other components of the pharmaceutical composition. Various purifiedtypes of BBI are disclosed in PCT International Application Publ. No. WO2013/10289 to Avraham Hershko, filed on Jan. 31, 2013, the contents ofwhich are incorporated herein by reference.

In other embodiments, the isolated BBI in the described methods andcompositions is a recombinant BBI, for example BBI produced by amicroorganism such as a bacterium that has been engineered to express itand subsequently isolated. In still other embodiments, the BBI is asynthetic BBI. An example of a synthetic BBI is BBI that has beenproduced in a cell-free apparatus such as a peptide synthesizer. Peptidesynthesizers, for example automated peptide synthesizers, are well knownin the art and are available commercially. Pharmaceutical compositionscomprising recombinant BBI are also provided herein. Pharmaceuticalcompositions comprising synthetic BBI are also provided herein.

In certain embodiments, the described BBI is the only protease inhibitorin the described methods and compositions. While lower-potency SBTIrequires an additional protease inhibitor, e.g. aprotinin, toefficiently protect certain therapeutic proteins in the human digestivetract, the described isolated BBI is, in some embodiments, capable ofreducing the need for additional protease inhibitors in this regard.

Methods for preparing KTI3 are described for example in PCTInternational Application Publ. No. WO 2013/102899 to Avraham Hershko,filed on Jan. 31, 2013, the contents of which are incorporated herein byreference.

KTI3 has Uniprot number P01070 (database accessed on Jan. 3, 2013). Arepresentative precursor sequence of KTI3 is:

(SEQ ID NO: 3) MKSTIFFLFL FCAFTTSYLP SAIADFVLDN EGNPLENGGTYYILSDITAF GGIRAAPTGN ERCPLTVVQS RNELDKGIGTIISSPYRIRF IAEGHPLSLK FDSFAVIMLC VGIPTEWSVVEDLPEGPAVK IGENKDAMDG WFRLERVSDD EFNNYKLVFCPQQAEDDKCG DIGISIDHDD GTRRLVVSKN KPLVVQFQKL DKESLAKKNH GLSRSE.

Of the above sequence, residues 1-24 are the signal peptide, 206-216 area propeptide, and the mature KTI3 chain is composed of residues 25-205(181 AA).

In certain embodiments, KTI3 is present in an amount between 25-125 mgper dosage unit and is present with a chymotrypsin inhibitor. In variousembodiments, KTI3 is present in an amount of 30-120 mg, 35-115 mg,40-110 mg, 45-105 mg, 50-100 mg, 55-95 mg, 60-90 mg, 65-85 mg, 70-80 mg,25-100 mg, 30-90 mg, 35-80 mg, 35-70 mg, 40-60 mg, 45-55 mg, 80-120 mg,85-115 mg, 90-100 mg, or 95-105 mg per dosage unit. All theaforementioned ranges are inclusive. In still other embodiments, theKTI3 is present in an amount of 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 75mg, 90 mg, 100 mg, 110 mg, or 120 mg per dosage unit.

In an additional aspect, a KTI3 utilized in the described methods andcompositions is at least 85% pure as measured, in various embodiments,by SDS-PAGE, Brilliant Blue staining, or imager quantitation.

In yet another aspect, the protein content of the KTI3 is greater than95% as measured by BCA assay.

In yet another aspect, the KTI3 contains less than 0.1% high-MWcontaminants, for example as assessed by SDS-PAGE and imagerquantitation.

In other embodiments, the isolated KTI3 has an anti-trypsin activity ofat least 0.8, 0.9, 1.0, 1.1, 1.2, or 1.3 mg. trypsin inhibited per mg.inhibitor. In other embodiments, the activity of the KTI3 is in therange of 0.8-1.8, 0.9-1.8, 1.0-1.8, 1.1-1.8, 1.2-1.8, or 1.3-1.8 mg.trypsin inhibited per mg. inhibitor. In more particular embodiments, theactivity of the KTI3 is 0.8-1.7 mg. trypsin inhibited per mg. inhibitor.In other embodiments, the activity is range of 0.8-1.4 mg. trypsininhibited per mg. inhibitor.

In other preferred embodiments, the isolated KTI is a recombinant KTI,for example KTI produced by a microorganism such as a bacterium that hasbeen engineered to express it. In still other preferred embodiments, theKTI is a synthetic KTI. An example of a synthetic KTI is KTI that hasbeen produced in a cell-free apparatus such as a peptide synthesizer.

Those skilled in the art will appreciate that each of the above purityrequirements, regarding its protein content, level of contaminants, orpotency, is typically assessed prior to the KTI3 being mixed with any ofthe other components of the pharmaceutical composition. Various purifiedtypes of KTI are disclosed in PCT International Application Publ. No. WO2013/10289 to Avraham Hershko, filed on Jan. 31, 2013, the contents ofwhich are incorporated herein by reference.

Other embodiments concern the ratio of the anti-chymotrypsin activitypresent in the described pharmaceutical composition to the anti-trypsinactivity of the composition. In some embodiments, this parameter isbetween 1.5:1 and 1:1. In more specific embodiments, the ratio may bebetween 1.4:1-1.1:1. In more specific embodiments, the ratio may bebetween 1.35:1-1.2:1.

In still other embodiments, the described purified SBTI, BBI, and/or KTIis present together with at least one of the described emulsifiers, insome embodiments in combination with at least one of the describednon-ionic detergents. Each described embodiment of the purified SBTI,BBI, and/or KTI may be freely combined with the described embodiments ofemulsifiers and/or non-ionic detergents.

Combinations of Protease Inhibitors

In certain embodiments, the pharmaceutical composition utilized in thedescribed methods and compositions comprises a combination of anisolated BBI and another protease inhibitor, for example a trypsininhibitor. In other embodiments, the pharmaceutical compositioncomprises a combination of an isolated KTI3 and another proteaseinhibitor. Those skilled in the art will appreciate in light of thepresent disclosure that a variety of protease inhibitors, includingthose mentioned hereinabove, may be utilized. In the case of proteaseinhibitors that are proteins, the size will typically be up to 100 kDa.

In other embodiments, the pharmaceutical composition utilized in thedescribed methods and compositions comprises both isolated BBI andisolated KTI, in more specific embodiments both isolated BBI andisolated KTI3. The terms “isolated KTI3 and “isolated BBI” as usedherein refers to a preparation enriched in the named component relativeto other component of SBTI (i.e. BBI or KTI3, respectively). In variousembodiments, the preparation of KTI utilized in the described methodsand compositions is at least 85% pure as assessed by SDS-PAGE, BrilliantBlue staining, or imager quantitation (e.g. according to the protocoldescribed herein). In other embodiments, the protein content of the KTIpreparation is greater than 95% by BCA assay. In the context of apharmaceutical composition, these values refer to characteristics of theKTI prior to its being mixed with one or more other components of thepharmaceutical composition. In other embodiments, the KTI preparationcontains 5% or less BBI as assessed by SDS-PAGE. In other embodiments,the KTI preparation contains less than 0.1% high-MW contaminants (inother words, substances having a MW of greater than 30,000).

In yet other embodiments, the weight/weight (w/w) ratio of BBI to KTI3in the pharmaceutical composition is between 1.5:1 and 2.5:1, in otherembodiments between 1.6:1 and 2.4:1, in other embodiments between 1.7:1and 2.3:1, in other embodiments between 1.8:1 and 2.2:1, in otherembodiments between 1.9:1 and 2.1:1, in other embodiments between 1.95:1and 2.05:1, in other embodiments 2:1.

In still more specific embodiments, the described methods andcompositions comprise the described BBI and KTI, in more specificembodiments BBI and KTI3, as the only protease inhibitors. In otherembodiments, the described methods and compositions comprise KTI andaprotinin, in more specific embodiments KTI3 and aprotinin, as the onlyprotease inhibitors. In other embodiments, isolated BBI, isolated KTI,and aprotinin are all present in the pharmaceutical composition.

In yet other embodiments, BBI and KTI3 are present in amounts of 50-100and 25-50 mg, respectively, per dosage form, and are the only proteaseinhibitors in the composition. In other embodiments, the amounts are55-90 and 25-45 mg, respectively; 60-90 and 25-45 mg, respectively;60-85 and 25-45 mg, respectively; 60-80 and 25-45 mg, respectively;65-75 and 30-40 mg, respectively; 68-72 and 33-37 mg, respectively; or70 and 35 mg, respectively. In still other embodiments, theaforementioned amounts of BBI and KTI3 are present together with anothertrypsin inhibitor, for example aprotinin.

In still other embodiments, the described combinations of proteaseinhibitors are present together with the described emulsifiers, in someembodiments in combination with the described non-ionic detergents. Eachdescribed combination of protease inhibitors may be freely combined withthe described embodiments of emulsifiers and/or non-ionic detergents.

Chelators of Divalent Cations

The chelator of divalent cations utilized in the described methods andcompositions is, in one embodiment, any physiologically acceptablecompound having a high affinity for at least one of calcium, magnesium,and manganese ions. In another embodiment, the chelator is selected fromthe group consisting of citrate or a salt thereof; ethylenediaminetetracetic acid (EDTA) or a salt thereof (for example disodium EDTA andcalcium disodium EDTA); EGTA (ethylene glycol tetraacetic acid) or asalt thereof; diethylene triamine pentaacetic acid (DTPA) or a saltthereof; and BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraaceticacid) or a salt thereof. In other embodiments, one of the above-listedchelators is utilized. In more specific embodiments, the chelator isEDTA.

Oils

Certain embodiments of pharmaceutical compositions and methods describedherein comprise a liquid phase, wherein one or more oils is utilized asthe basis of the liquid phase. In certain embodiments, the oil may beany physiologically acceptable oil that is liquid at ambienttemperature.

Reference herein to components which are “in an oil” or “in a liquid”means that each of the named components are dissolved, suspended, and/oremulsified in an oil or oil phase. Reference to “oil-based” compositionsmeans that all the solid components of the compositions (i.e. thecomponents that are usually provided as a solid in a tableting process)are dissolved, suspended, and/or emulsified in an oil or oil phase. Insome embodiments, an oil phase is the only liquid component of thepharmaceutical composition. In more specific embodiments, the oil phaseis the only component of the pharmaceutical composition, other than thecapsule and optional other coatings.

In other embodiments, the oil-based liquid phase of the pharmaceuticalcomposition is water-free, or in other embodiments is alcohol-free, orin other embodiments is both alcohol-free and water-free. In otherembodiments, the oil or mixture of oils are the only liquid componentsof the oil phase other than one or more optional emulsifiers orsurfactants. In still other embodiments, the oil or mixture of oils arethe only liquid components of the oil phase. In yet other embodiments,the oil phase consists of the oil or mixture of oils and the solidcomponents dissolved, suspended, and/or emulsified therein. In yet otherembodiments, the oil phase consists of the oil or mixture of oils andone or more therapeutic proteins, one or more protease inhibitors, and achelator of divalent cations, all of which are dissolved, suspended,and/or emulsified therein. In yet other embodiments, the oil phaseconsists of the oil or mixture of oils; one or more therapeuticproteins, one or more protease inhibitors, and a chelator of divalentcations, all of which are dissolved, suspended, and/or emulsifiedtherein; and one or more optional emulsifiers or surfactants. Traceliquid components, such as water absorbed from the atmosphere, is notconsidered to be present for purposes of classifying a composition as“water-free”, having an oil or mixture of oils as the “only liquidcomponents”, or the like.

In more specific embodiments, the oil comprises an omega-3 fatty acid.In other embodiments, the omega-3 fatty acid is an omega-3polyunsaturated fatty acid. In another embodiment, the omega-3 fattyacid is DHA, an omega-3, polyunsaturated, 22-carbon fatty acid alsoreferred to as 4, 7, 10, 13, 16, 19-docosahexaenoic acid. In anotherembodiment, the omega-3 fatty acid is linolenic acid (9, 12,15-octadecatrienoic acid). In another embodiment, the omega-3 fatty acidis stearidonic acid (6, 9, 12, 15-octadecatetraenoic acid). In anotherembodiment, the omega-3 fatty acid is eicosatrienoic acid (ETA; 11, 14,17-eicosatrienoic acid). In another embodiment, the omega-3 fatty acidis eicsoatetraenoic acid (8, 11, 14, 17-eicosatetraenoic acid). In oneembodiment, the omega-3 fatty acid is eicosapentaenoic acid (EPA; 5, 8,11, 14, 17-eicosapentaenoic acid). In another embodiment, the omega-3fatty acid is eicosahexaenoic acid (also referred to as 5, 7, 9, 11, 14,17-eicosahexaenoic acid). In another embodiment, the omega-3 fatty acidis docosapentaenoic acid (DPA; 7, 10, 13, 16, 19-docosapenatenoic acid).In another embodiment, the omega-3 fatty acid is tetracosahexaenoic acid(6, 9, 12, 15, 18, 21-tetracosahexaenoic acid).

In other embodiments, the oil is a naturally-occurring oil comprising anomega-3 fatty acid. In other embodiments, the oil is anaturally-occurring oil that is generally recognized as safe (GRAS). Inother embodiments, the oil is a naturally-occurring GRAS vegetable oil.In certain embodiments, the oil may be selected from cardamom oil,carrot seed oil, Roman chamomile oil, coriander oil, black cumin oil,goldenrod oil, mandarin oil, lemon balm oil, sandalwood oil, spearmintoil, olive oil, flaxseed oil, sesame oil, avocado oil, walnut oil,canola oil, cottonseed oil, corn oil, safflower oil, sunflower oil,soybean oil, grapeseed oil, almond oil, and fish oil, non-limitingexamples of which are salmon oil and tuna oil. In more specificembodiments, the oil may be selected from olive oil, flaxseed oil,sesame oil, avocado oil, walnut oil, canola oil, and fish oil. In stillmore specific embodiments, the oil is selected from fish oil, canolaoil, and flaxseed oil. Alternatively, the oil is selected from the groupconsisting of fish oil, canola oil, flaxseed oil, algal oil and hempseed oil. In more specific embodiments, the oil is a fish oil. Severaltypes of fish oil have been tested in the compositions described hereinand have all been found to work equally well.

Neurodegenerative Disorders

It will be appreciated by those skilled in the art GLP-1 can have atherapeutic effect on neurodegenerative disorders independently of itseffects on NAFLD. Those skilled in the art will appreciate thatneurodegenerative disorders can be diagnosed by a number of knownmethods, including MRI (magnetic resonance imaging) of the centralnervous system.

Provided herein, in another embodiment, is a pharmaceutical compositionfor treating, or in another aspect reducing the incidence of,Alzheimer's disease, said pharmaceutical composition comprising insulin,a GLP-1 analogue, at least one protease inhibitor, and a chelator ofdivalent cations. In another embodiment, the pharmaceutical compositioncomprises a GLP-1 analogue, at least one protease inhibitor, and achelator of divalent cations. In certain embodiments, the pharmaceuticalcomposition comprises one of the oil-based liquid formulations describedherein, wherein the pharmaceutical composition may further comprise, insome embodiments, a capsule and/or coating that resists degradation inthe stomach. In other embodiments, the pharmaceutical compositioncomprises one of the solid formulations described herein. In certainembodiments, the pharmaceutical composition is administered to thesubject for an extended time.

Still another aspect provides a method of treating, or in another aspectreducing the incidence of, Alzheimer's disease, the method comprisingthe step of administering to a subject a pharmaceutical compositiondescribed hereinabove, thereby treating or reducing the incidence ofAlzheimer's disease.

Provided herein, in another embodiment, is a pharmaceutical compositionfor treating, or in another aspect reducing the incidence of,Parkinson's disease, said pharmaceutical composition comprising insulin,a GLP-1 analogue, at least one protease inhibitor, and a chelator ofdivalent cations. In another embodiment, the pharmaceutical compositioncomprises a GLP-1 analogue, at least one protease inhibitor, and achelator of divalent cations. In certain embodiments, the pharmaceuticalcomposition comprises one of the oil-based liquid formulations describedherein, wherein the pharmaceutical composition may further comprise, insome embodiments, a capsule and/or coating that resists degradation inthe stomach. In other embodiments, the pharmaceutical compositioncomprises one of the solid formulations described herein. In certainembodiments, the pharmaceutical composition is administered to thesubject for an extended time.

Still another aspect provides a method of treating, or in another aspectreducing the incidence of, Parkinson's disease, the method comprisingthe step of administering to a subject a pharmaceutical compositiondescribed hereinabove, thereby treating or reducing the incidence ofParkinson's disease.

Provided herein, in another embodiment, is a pharmaceutical compositionfor treating, or in another aspect reducing the incidence of,Huntington's disease, said pharmaceutical composition comprisinginsulin, a GLP-1 analogue, at least one protease inhibitor, and achelator of divalent cations. In another embodiment, the pharmaceuticalcomposition comprises a GLP-1 analogue, at least one protease inhibitor,and a chelator of divalent cations. In certain embodiments, thepharmaceutical composition comprises one of the oil-based liquidformulations described herein, wherein the pharmaceutical compositionmay further comprise, in some embodiments, a capsule and/or coating thatresists degradation in the stomach. In other embodiments, thepharmaceutical composition comprises one of the solid formulationsdescribed herein. In certain embodiments, the pharmaceutical compositionis administered to the subject for an extended time.

Still another aspect provides a method of treating, or in another aspectreducing the incidence of, Huntington's disease, the method comprisingthe step of administering to a subject a pharmaceutical compositiondescribed hereinabove, thereby treating or reducing the incidence ofHuntington's disease.

Provided herein, in another embodiment, is a pharmaceutical compositionfor treating, or in another aspect reducing the incidence of, ALS, saidpharmaceutical composition comprising insulin, a GLP-1 analogue, atleast one protease inhibitor, and a chelator of divalent cations. Inanother embodiment, the pharmaceutical composition comprises a GLP-1analogue, at least one protease inhibitor, and a chelator of divalentcations. In certain embodiments, the pharmaceutical compositioncomprises one of the oil-based liquid formulations described herein,wherein the pharmaceutical composition may further comprise, in someembodiments, a capsule and/or coating that resists degradation in thestomach. In other embodiments, the pharmaceutical composition comprisesone of the solid formulations described herein. In certain embodiments,the pharmaceutical composition is administered to the subject for anextended time.

Still another aspect provides a method of treating, or in another aspectreducing the incidence of, ALS, the method comprising the step ofadministering to a subject a pharmaceutical composition describedhereinabove, thereby treating or reducing the incidence of ALS.

Provided herein, in another embodiment, is a pharmaceutical compositionfor treating, or in another aspect reducing the incidence of, traumaticbrain injury (TBI), said pharmaceutical composition comprising insulin,a GLP-1 analogue, at least one protease inhibitor, and a chelator ofdivalent cations. In another embodiment, the pharmaceutical compositioncomprises a GLP-1 analogue, at least one protease inhibitor, and achelator of divalent cations. In certain embodiments, the pharmaceuticalcomposition comprises one of the oil-based liquid formulations describedherein, wherein the pharmaceutical composition may further comprise, insome embodiments, a capsule and/or coating that resists degradation inthe stomach. In other embodiments, the pharmaceutical compositioncomprises one of the solid formulations described herein. In certainembodiments, the pharmaceutical composition is administered to thesubject for an extended time.

Still another aspect provides a method of treating, or in another aspectreducing the incidence of, TBI, the method comprising the step ofadministering to a subject a pharmaceutical composition describedhereinabove, thereby treating or reducing the incidence of TBI.

Provided herein, in another embodiment, is a pharmaceutical compositionfor treating, or in another aspect reducing the incidence of, mooddisorders, said pharmaceutical composition comprising insulin, a GLP-1analogue, at least one protease inhibitor, and a chelator of divalentcations. In another embodiment, the pharmaceutical composition comprisesa GLP-1 analogue, at least one protease inhibitor, and a chelator ofdivalent cations. In certain embodiments, the pharmaceutical compositioncomprises one of the oil-based liquid formulations described herein,wherein the pharmaceutical composition may further comprise, in someembodiments, a capsule and/or coating that resists degradation in thestomach. In other embodiments, the pharmaceutical composition comprisesone of the solid formulations described herein. In certain embodiments,the pharmaceutical composition is administered to the subject for anextended time.

Still another aspect provides a method of treating, or in another aspectreducing the incidence of, mood disorders, the method comprising thestep of administering to a subject a pharmaceutical compositiondescribed hereinabove, thereby treating or reducing the incidence ofmood disorders.

Provided herein, in another embodiment, is a pharmaceutical compositionfor reducing tissue damage from cerebral stroke, said pharmaceuticalcomposition comprising insulin, a GLP-1 analogue, at least one proteaseinhibitor, and a chelator of divalent cations. In another embodiment,the pharmaceutical composition comprises a GLP-1 analogue, at least oneprotease inhibitor, and a chelator of divalent cations. In certainembodiments, the pharmaceutical composition comprises one of theoil-based liquid formulations described herein, wherein thepharmaceutical composition may further comprise, in some embodiments, acapsule and/or coating that resists degradation in the stomach. In otherembodiments, the pharmaceutical composition comprises one of the solidformulations described herein. In certain embodiments, thepharmaceutical composition is administered to the subject for anextended time.

Still another aspect provides a method of reducing tissue damage fromcerebral stroke, the method comprising the step of administering to asubject a pharmaceutical composition described hereinabove, therebyreducing tissue damage from cerebral stroke.

Provided herein, in another embodiment, is a pharmaceutical compositionfor treating, or in another aspect reducing the incidence of, retinaldegeneration, said pharmaceutical composition comprising insulin, aGLP-1 analogue, at least one protease inhibitor, and a chelator ofdivalent cations. In another embodiment, the pharmaceutical compositioncomprises a GLP-1 analogue, at least one protease inhibitor, and achelator of divalent cations. In certain embodiments, the pharmaceuticalcomposition comprises one of the oil-based liquid formulations describedherein, wherein the pharmaceutical composition may further comprise, insome embodiments, a capsule and/or coating that resists degradation inthe stomach. In other embodiments, the pharmaceutical compositioncomprises one of the solid formulations described herein. In certainembodiments, the pharmaceutical composition is administered to thesubject for an extended time.

Still another aspect provides a method of treating, or in another aspectreducing the incidence of, retinal degeneration, the method comprisingthe step of administering to a subject a pharmaceutical compositiondescribed hereinabove, thereby treating or reducing the incidence ofretinal degeneration.

Provided herein, in another embodiment, is a pharmaceutical compositionfor treating, or in another aspect reducing the incidence of, peripheralneuropathy (PN), said pharmaceutical composition comprising insulin, aGLP-1 analogue, at least one protease inhibitor, and a chelator ofdivalent cations. In another embodiment, the pharmaceutical compositioncomprises a GLP-1 analogue, at least one protease inhibitor, and achelator of divalent cations. In certain embodiments, the pharmaceuticalcomposition comprises one of the oil-based liquid formulations describedherein, wherein the pharmaceutical composition may further comprise, insome embodiments, a capsule and/or coating that resists degradation inthe stomach. In other embodiments, the pharmaceutical compositioncomprises one of the solid formulations described herein. In certainembodiments, the pharmaceutical composition is administered to thesubject for an extended time.

Still another aspect provides a method of treating, or in another aspectreducing the incidence of, PN, the method comprising the step ofadministering to a subject a pharmaceutical composition describedhereinabove, thereby treating or reducing the incidence of PN.

Representative Specific Formulations

In certain embodiments, a formulation utilized in the described methodsand compositions is a liquid formulation that comprises insulin, a GLP-1analogue, and at least one trypsin inhibitor, all of which are in anoil, for example dissolved, suspended, and/or emulsified in the oil. Inmore specific embodiments, the GLP-1 analogue may be exenatide. In otherembodiments, the described liquid formulation comprises insulin, a GLP-1analogue, and at least one chymotrypsin inhibitor. In still otherembodiments, the described liquid formulation comprises insulin, a GLP-1analogue, at least one trypsin inhibitor, and at least one chymotrypsininhibitor. In still other embodiments, the described liquid formulationcomprises insulin, a GLP-1 analogue, one trypsin inhibitor, and onechymotrypsin inhibitor. In more specific embodiments, the proteaseinhibitors are SBTI and aprotinin. Alternatively, purified KTI3 andaprotinin are utilized. In still other embodiments, purified BBI is theonly protease inhibitor present. In other embodiments, a chelator ofdivalent cations is also present in the liquid formulation, which is, inmore specific embodiments, EDTA. Alternatively or in addition, theliquid formulation is in a gelatin capsule. In more specificembodiments, the gelatin capsule is coated with an enteric coating.

Reference herein to a trypsin inhibitor in combination with achymotrypsin inhibitor encompasses cases where a trypsin inhibitor suchas KTI3 is present together with an inhibitor of both chymotrypsin andtrypsin.

In certain embodiments, a formulation utilized in the described methodsand compositions is a liquid formulation that comprises insulin and atleast one trypsin inhibitor, both/all of which are in an oil, forexample dissolved, suspended, and/or emulsified in the oil. In otherembodiments, the described liquid formulation comprises insulin and atleast one chymotrypsin inhibitor. In still other embodiments, thedescribed liquid formulation comprises insulin, at least one trypsininhibitor, and at least one chymotrypsin inhibitor. In still otherembodiments, the described liquid formulation comprises insulin, onetrypsin inhibitor, and one chymotrypsin inhibitor. In more specificembodiments, the protease inhibitors are SBTI and aprotinin.Alternatively, purified KTI3 and aprotinin are utilized. In still otherembodiments, purified BBI is the only protease inhibitor present. Inother embodiments, a chelator of divalent cations is also present in theliquid formulation, which is, in more specific embodiments, EDTA.Alternatively or in addition, the liquid formulation is in a gelatincapsule. In more specific embodiments, the gelatin capsule is coatedwith an enteric coating.

In certain embodiments, a formulation utilized in the described methodsand compositions is a liquid formulation that comprises a GLP-1 analogueand at least one trypsin inhibitor, both/all of which are in an oil, forexample dissolved, suspended, and/or emulsified in the oil. In morespecific embodiments, the GLP-1 analogue may be exenatide. In otherembodiments, the described liquid formulation comprises a GLP-1 analogueand at least one chymotrypsin inhibitor. In still other embodiments, thedescribed liquid formulation comprises a GLP-1 analogue, at least onetrypsin inhibitor, and at least one chymotrypsin inhibitor. In stillother embodiments, the described liquid formulation comprises a GLP-1analogue, one trypsin inhibitor, and one chymotrypsin inhibitor. In morespecific embodiments, the protease inhibitors are SBTI and aprotinin.Alternatively, purified KTI3 and aprotinin are utilized. In still otherembodiments, purified BBI is the only protease inhibitor present. Inother embodiments, a chelator of divalent cations is also present in theliquid formulation, which is, in more specific embodiments, EDTA.Alternatively or in addition, the liquid formulation is in a gelatincapsule. In more specific embodiments, the gelatin capsule is coatedwith an enteric coating.

In other embodiments, a liquid formulation utilized in the describedmethod or composition comprises insulin, exenatide, a surfactant, EDTA,SBTI, aprotinin, and oil. In some embodiments, the surfactant referredto in this paragraph may be Gelucire 44/14. In other embodiments, theliquid formulation consists essentially of insulin, exenatide, asurfactant, EDTA, SBTI, aprotinin, and oil. “Consists essentially of” inthis and the below paragraphs indicates that the liquid formulation doesnot contain any other components that appreciably affect itsphysiological characteristics. In other embodiments, the liquidformulation consists of insulin, exenatide, a surfactant, EDTA, SBTI,aprotinin, and oil. In other, even more specific embodiments, theamounts of insulin, exenatide, EDTA, SBTI, aprotinin, and oil per dosageform are 8-16 mg, 150-300 mcg, 100-200 mg, 50-100 mg, 20-30 mg, and0.4-0.7 ml, respectively, and the amount of surfactant, e.g. Gelucire44/14, is 8-16%. In still more specific embodiments, the amounts ofinsulin, exenatide, EDTA, SBTI, aprotinin, and oil per dosage form are8-16 mg, 150-300 mcg, 150 mg, 65-85 mg, 22-26 mg, and 0.5-0.7 ml,respectively, and the amount of surfactant, e.g. Gelucire 44/1444/14 is8-16%. An even more specific embodiment comprises 75 mg SBTI and 24 mgaprotinin. In other embodiments, the above composition further comprisespolysorbate 80. In some embodiments, the polysorbate 80 constitutes3-10% weight/weight inclusive of the oil-based liquid formulation. Incertain embodiments, all the aforementioned ingredients (other than theoil) are all provided in the oil, for example dissolved or suspended inthe oil, which is, in some embodiments, one of the oils mentionedherein. Alternatively or in addition, the above composition is coated bya coating that resists degradation in the stomach. Alternatively or inaddition, purified KTI3 is present in place of SBTI in one of theamounts mentioned above. In various embodiments, purified BBI is theonly protease inhibitor present and is present in an amount of 50-200mg, in other embodiments 75-200 mg, in other embodiments 75-180 mg, inother embodiments 75-150 mg, in other embodiments 75-125 mg, in otherembodiments 90-110 mg, in other embodiments 100-150 mg, in otherembodiments 110-140 mg, in other embodiments 120-130 mg, in otherembodiments 120-160 mg, in other embodiments 130-150 mg, in otherembodiments 75 mg, in other embodiments 100 mg, in other embodiments 125mg, in other embodiments 150 mg, in other embodiments 175 mg, in otherembodiments 200 mg.

In more specific embodiments, a liquid formulation utilized in thedescribed method or composition comprises insulin; exenatide; aself-emulsifying component, for example a component provided as amixture of (a) a monoacylglycerol, a diacylglycerol, a triacylglycerol,or a mixture thereof; and (b) a PEG ester of a fatty acid; EDTA; SBTI;aprotinin; and oil. In other embodiments, the liquid formulationconsists essentially of insulin, exenatide, a self-emulsifyingcomponent, EDTA, SBTI, aprotinin, and oil. In other embodiments, theliquid formulation consists of insulin, exenatide, a self-emulsifyingcomponent, EDTA, SBTI, aprotinin, and oil. In other, even more specificembodiments, the amounts of insulin, exenatide, EDTA, SBTI, aprotinin,and oil per dosage form are 8-16 mg, 150-300 mcg, 100-200 mg, 50-100 mg,20-30 mg, and 0.4-0.7 ml, respectively, and the amount ofself-emulsifying component is 8-16%. In still more specific embodiments,the amounts of insulin, exenatide, EDTA, SBTI, aprotinin, and oil perdosage form are 8-16 mg, 150-300 mcg, 150 mg, 65-85 mg, 22-26 mg, and0.5-0.7 ml, respectively, and the amount of self-emulsifying componentis 8-16%. An even more specific embodiment comprises 75 mg SBTI and 24mg aprotinin. In other embodiments, the above composition furthercomprises polysorbate 80. In certain embodiments, the polysorbate 80constitutes 3-10% (inclusive) weight/weight of the oil-based liquidformulation. In certain embodiments, all the aforementioned ingredientsother than the oil are provided in the oil, for example dissolved orsuspended in the oil, which is, in some embodiments, one of the oilsmentioned herein. Alternatively or in addition, the above composition iscoated by a coating that resists degradation in the stomach.Alternatively or in addition, purified KTI3 is present in place of SBTIin one of the amounts mentioned above. In other embodiments, purifiedBBI is present in one of the amounts mentioned above for SBTI and is theonly protease inhibitor present.

In other embodiments, a liquid formulation utilized in the describedmethod or composition comprises exenatide, a surfactant, EDTA, SBTI,aprotinin, and oil. In some embodiments, the surfactant referred to inthis paragraph may be Gelucire 44/14. In other embodiments, the liquidformulation consists essentially of exenatide, a surfactant, EDTA, SBTI,aprotinin, and oil. In other embodiments, the liquid formulationconsists of exenatide, a surfactant, EDTA, SBTI, aprotinin, and oil. Inother, even more specific embodiments, the amounts of exenatide, EDTA,SBTI, aprotinin, and oil per dosage form are 150-300 mcg, 100-200 mg,50-100 mg, 20-30 mg, and 0.4-0.7 ml, respectively, and the amount of asurfactant, e.g. Gelucire 44/14 is 8-16%. In still more specificembodiments, the amounts of exenatide, EDTA, SBTI, aprotinin, and oilper dosage form are 150-300 mcg, 150 mg, 65-85 mg, 22-26 mg, and 0.5-0.7ml, respectively, and the amount of surfactant, e.g. Gelucire 44/1444/14is 8-16%. An even more specific embodiment comprises 75 mg SBTI and 24mg aprotinin. In other embodiments, the above composition furthercomprises polysorbate 80. In some embodiments, the polysorbate 80constitutes 3-10% weight/weight inclusive of the oil-based liquidformulation. In certain embodiments, all the aforementioned ingredientsother than the oil are provided in the oil, for example dissolved orsuspended in the oil, which is, in some embodiments, one of the oilsmentioned herein. Alternatively or in addition, the above composition iscoated by a coating that resists degradation in the stomach.Alternatively or in addition, purified KTI3 is present in place of SBTIin one of the amounts mentioned above. In various embodiments, purifiedBBI is the only protease inhibitor present and is present in an amountof 50-200 mg, in other embodiments 75-200 mg, in other embodiments75-180 mg, in other embodiments 75-150 mg, in other embodiments 75-125mg, in other embodiments 90-110 mg, in other embodiments 100-150 mg, inother embodiments 110-140 mg, in other embodiments 120-130 mg, in otherembodiments 120-160 mg, in other embodiments 130-150 mg, in otherembodiments 75 mg, in other embodiments 100 mg, in other embodiments 125mg, in other embodiments 150 mg, in other embodiments 175 mg, in otherembodiments 200 mg.

In still more specific embodiments, a liquid formulation utilized in thedescribed method or composition comprises exenatide, a self-emulsifyingcomponent, EDTA, SBTI, aprotinin, and oil. In other embodiments, theliquid formulation consists essentially of exenatide, a self-emulsifyingcomponent, EDTA, SBTI, aprotinin, and oil. In other embodiments, theliquid formulation consists of exenatide, a self-emulsifying component,EDTA, SBTI, aprotinin, and oil. In other, even more specificembodiments, the amounts of exenatide, EDTA, SBTI, aprotinin, and oilper dosage form are 150-300 mcg, 100-200 mg, 50-100 mg, 20-30 mg, and0.4-0.7 ml, respectively, and the amount of self-emulsifying componentis 8-16%. In still more specific embodiments, the amounts of exenatide,EDTA, SBTI, aprotinin, and oil per dosage form are 150-300 mcg, 150 mg,65-85 mg, 22-26 mg, and 0.5-0.7 ml, respectively, and the amount ofself-emulsifying component is 8-16%. An even more specific embodimentcomprises 75 mg SBTI and 24 mg aprotinin. In other embodiments, theabove composition further comprises polysorbate 80. In certainembodiments, the polysorbate 80 constitutes 3-10% weight/weightinclusive of the oil-based liquid formulation. In certain embodiments,all the aforementioned ingredients other than the oil are provided inthe oil, for example dissolved or suspended in the oil, which is, insome embodiments, one of the oils mentioned herein. Alternatively or inaddition, the above composition is coated by a coating that resistsdegradation in the stomach. Alternatively or in addition, purified KTI3is present in place of SBTI in one of the amounts mentioned above. Invarious embodiments, purified BBI is the only protease inhibitor presentand is present in an amount of 50-200 mg, in other embodiments 75-200mg, in other embodiments 75-180 mg, in other embodiments 75-150 mg, inother embodiments 75-125 mg, in other embodiments 90-110 mg, in otherembodiments 100-150 mg, in other embodiments 110-140 mg, in otherembodiments 120-130 mg, in other embodiments 120-160 mg, in otherembodiments 130-150 mg, in other embodiments 75 mg, in other embodiments100 mg, in other embodiments 125 mg, in other embodiments 150 mg, inother embodiments 175 mg, in other embodiments 200 mg.

In other embodiments, a liquid formulation utilized in the describedmethod or composition comprises insulin, Gelucire 44/14, EDTA, SBTI,aprotinin, and oil. In other embodiments, the liquid formulationconsists essentially of insulin, Gelucire 44/14, EDTA, SBTI, aprotinin,and oil. In other embodiments, the liquid formulation consists ofinsulin, Gelucire 44/14, EDTA, SBTI, aprotinin, and oil. In other, evenmore specific embodiments, the amounts of insulin, EDTA, SBTI,aprotinin, and oil per dosage form are 8-16 mg, 100-200 mg, 50-100 mg,20-30 mg, and 0.4-0.7 ml, respectively, and the amount of Gelucire 44/14is 8-16%. In still more specific embodiments, the amounts of insulin,EDTA, SBTI, aprotinin, and oil per dosage form are 8-16 mg, 150 mg,65-85 mg, 22-26 mg, and 0.5-0.7 ml, respectively, and the amount ofGelucire 44/14 is 8-16%. An even more specific embodiment comprises 75mg SBTI and 24 mg aprotinin. In other embodiments, the above compositionfurther comprises polysorbate 80. In some embodiments, the polysorbate80 constitutes 3-10% weight/weight inclusive of the oil-based liquidformulation. In certain embodiments, all the aforementioned ingredientsother than the oil are provided in the oil, for example dissolved orsuspended in the oil, which is, in some embodiments, one of the oilsmentioned herein. Alternatively or in addition, the above composition iscoated by a coating that resists degradation in the stomach.Alternatively or in addition, purified KTI3 is present in place of SBTIin one of the amounts mentioned above. In various embodiments, purifiedBBI is the only protease inhibitor present and is present in an amountof 50-200 mg, in other embodiments 75-200 mg, in other embodiments75-180 mg, in other embodiments 75-150 mg, in other embodiments 75-125mg, in other embodiments 90-110 mg, in other embodiments 100-150 mg, inother embodiments 110-140 mg, in other embodiments 120-130 mg, in otherembodiments 120-160 mg, in other embodiments 130-150 mg, in otherembodiments 75 mg, in other embodiments 100 mg, in other embodiments 125mg, in other embodiments 150 mg, in other embodiments 175 mg, in otherembodiments 200 mg.

In still more specific embodiments, a liquid formulation utilized in thedescribed method or composition comprises insulin, a self-emulsifyingcomponent, EDTA, SBTI, aprotinin, and oil. In other embodiments, theliquid formulation consists essentially of insulin, a self-emulsifyingcomponent, EDTA, SBTI, aprotinin, and oil. In other embodiments, theliquid formulation consists of insulin, a self-emulsifying component,EDTA, SBTI, aprotinin, and oil. In other, even more specificembodiments, the amounts of insulin, EDTA, SBTI, aprotinin, and oil perdosage form are 8-16 mg, 100-200 mg, 50-100 mg, 20-30 mg, and 0.4-0.7ml, respectively, and the amount of self-emulsifying component is 8-16%.In still more specific embodiments, the amounts of insulin, EDTA, SBTI,aprotinin, and oil per dosage form are 8-16 mg, 150 mg, 65-85 mg, 22-26,and 0.5-0.7 ml, respectively, and the amount of self-emulsifyingcomponent is 8-16%. An even more specific embodiment comprises 75 mgSBTI and 24 mg aprotinin. In other embodiments, the above compositionfurther comprises polysorbate 80. In certain embodiments, thepolysorbate 80 constitutes 3-10% weight/weight inclusive of theoil-based liquid formulation. In certain embodiments, all theaforementioned ingredients other than the oil are provided in the oil,for example dissolved or suspended in the oil, which is, in someembodiments, one of the oils mentioned herein. Alternatively or inaddition, the above composition is coated by a coating that resistsdegradation in the stomach. Alternatively or in addition, purified KTI3is present in place of SBTI in one of the amounts mentioned above. Invarious embodiments, purified BBI is the only protease inhibitor presentand is present in an amount of 50-200 mg, in other embodiments 75-200mg, in other embodiments 75-180 mg, in other embodiments 75-150 mg, inother embodiments 75-125 mg, in other embodiments 90-110 mg, in otherembodiments 100-150 mg, in other embodiments 110-140 mg, in otherembodiments 120-130 mg, in other embodiments 120-160 mg, in otherembodiments 130-150 mg, in other embodiments 75 mg, in other embodiments100 mg, in other embodiments 125 mg, in other embodiments 150 mg, inother embodiments 175 mg, in other embodiments 200 mg.

“Weight/weight” percentages referred to herein in the context ofemulsifiers and detergents utilize the amount of oil base in theformulation, for example fish oil, as the denominator; thus, 60 mg ofGelucire (for example) in 500 mg oil is considered as 12% w/w,regardless of the weight of the other components. Similarly, 50 mg.Tween-80 mixed with 500 mg oil is considered as 10% Tween-80.

In other embodiments, a liquid formulation utilized in the describedmethod or composition is water-free. If more than one liquid formulationis present, for example in a multi-component composition, each liquidformulation may be water-free. “Water-free” refers, in certainembodiments, to a formulation into which no aqueous components have beenintentionally added. It does not preclude the presence of trace amountsof water that have been absorbed from the atmosphere into the componentsthereof. In another embodiment, the liquid formulation is free ofaqueous components. If more than one liquid formulation is present, forexample in a multi-component composition, each liquid formulation may befree of aqueous components. In yet other embodiments, one or more oilsselected from selected from olive oil, flaxseed oil, sesame oil, avocadooil, walnut oil, canola oil, and fish oil are the only liquid componentsof each of the one or more liquid formulations. In yet anotherembodiment, fish oil is the only liquid component of each of the one ormore liquid formulations. In still another embodiments, the only liquidcomponent of the one or more liquid formulations is selected from thegroup consisting of fish oil, canola oil, and flaxseed oil.

In other embodiments, a formulation utilized in the described methodsand compositions is a solid formulation that comprises insulin, a GLP-1analogue, EDTA, and at least one trypsin inhibitor. In more specificembodiments, the GLP-1 analogue may be exenatide. In other embodiments,the formulation comprises insulin, a GLP-1 analogue, EDTA, and at leastone chymotrypsin inhibitor. In other embodiments, the formulationcomprises insulin, a GLP-1 analogue, EDTA, at least one trypsininhibitor, and at least one chymotrypsin inhibitor. In otherembodiments, the formulation comprises insulin, a GLP-1 analogue, EDTA,one trypsin inhibitor, and one chymotrypsin inhibitor. In more specificembodiments, the protease inhibitors are SBTI and aprotinin.Alternatively, purified KTI3 and aprotinin are utilized. In variousembodiments, purified BBI is the only protease inhibitor present and ispresent in an amount of 50-200 mg, in other embodiments 75-200 mg, inother embodiments 75-180 mg, in other embodiments 75-150 mg, in otherembodiments 75-125 mg, in other embodiments 90-110 mg, in otherembodiments 100-150 mg, in other embodiments 110-140 mg, in otherembodiments 120-130 mg, in other embodiments 120-160 mg, in otherembodiments 130-150 mg, in other embodiments 75 mg, in other embodiments100 mg, in other embodiments 125 mg, in other embodiments 150 mg, inother embodiments 175 mg, in other embodiments 200 mg. Alternatively orin addition, the solid formulation is coated with an enteric coating.

The term “solid formulations” as used herein relates to tablets,capsules, and other dosage forms having a non-liquid structurethroughout the dosage form. In certain, non-limiting embodiments, thetherapeutic peptide(s), EDTA, and protease inhibitor(s), all incrystalline form, or in other embodiments in amorphous form, arecombined and subjected to a tableting process known in the art.

In other embodiments, a formulation utilized in the described methodsand compositions is a solid formulation that comprises a GLP-1 analogue,EDTA, and at least one trypsin inhibitor. In more specific embodiments,the GLP-1 analogue may be exenatide. In other embodiments, theformulation comprises a GLP-1 analogue, EDTA, and at least onechymotrypsin inhibitor. In other embodiments, the formulation comprisesa GLP-1 analogue, EDTA, at least one trypsin inhibitor, and at least onechymotrypsin inhibitor. In other embodiments, the formulation comprisesa GLP-1 analogue, EDTA, one trypsin inhibitor, and one chymotrypsininhibitor. In more specific embodiments, the protease inhibitors areSBTI and aprotinin. Alternatively, purified KTI3 and aprotinin areutilized. In still other embodiments, purified BBI is the only proteaseinhibitor present. Alternatively or in addition, the liquid formulationis in a gelatin capsule. In more specific embodiments, the gelatincapsule is coated with an enteric coating.

In other embodiments, a formulation utilized in the described methodsand compositions is a solid formulation that comprises insulin, EDTA,and at least one trypsin inhibitor. In other embodiments, theformulation comprises insulin, EDTA, and at least one chymotrypsininhibitor. In other embodiments, the formulation comprises insulin,EDTA, at least one trypsin inhibitor, and at least one chymotrypsininhibitor. In other embodiments, the formulation comprises insulin,EDTA, one trypsin inhibitor, and one chymotrypsin inhibitor. In morespecific embodiments, the protease inhibitors are SBTI and aprotinin.Alternatively, purified KTI3 and aprotinin are utilized. In still otherembodiments, purified BBI is the only protease inhibitor present.Alternatively or in addition, the liquid formulation is in a gelatincapsule. In more specific embodiments, the gelatin capsule is coatedwith an enteric coating.

Diabetes and Other Metabolic Disorders

Also provided herein is a solid pharmaceutical formulation describedherein for treating diabetes mellitus, which is in some embodiments typeI DM and is in other embodiments type II DM. Also provided is a methodfor treating diabetes mellitus by administering to a subject a solidpharmaceutical formulation described herein. In certain embodiments, thepharmaceutical composition is administered to the subject one or moretimes per day, depending on blood sugar levels. In other embodiments,the pharmaceutical composition is administered to the subject for anextended time.

In other embodiments is provided a solid pharmaceutical formulationdescribed herein, comprising, in various embodiments, insulin, a GLP-1analogue, or a combination thereof as the active ingredient, forinhibiting or reducing the development of type II diabetes mellitus(T2DM) in a subject at risk thereof. In certain embodiments, the subjectat risk is considered “pre-diabetic” and/or exhibits impaired glucosetolerance (IGT). IGT is typically considered to be present when fastingplasma glucose (FPG) levels are between 100-126 milligrams per deciliter(mg/dl) and/or 2-hour values in the oral glucose tolerance test (OGTT)are between 140-200 mg/dl; while T2DM is considered to be present basedupon a FPG value of >126 or a 2-hour OGTT value of >200 mg/dl. However,those skilled in the art will appreciate that the exact standards fordetermining IGT and T2DM are not critical, and other tests, e.g. thefrequently sampled intravenous glucose tolerance test (FSIVGTT) andHbA1C levels, may be utilized. In other embodiments, the subject at riskexhibits insulin resistance, obesity, and/or a family history ofdiabetes. In certain embodiments, the pharmaceutical composition isadministered for an extended time. In more specific embodiments, thepharmaceutical composition may be administered for an extended timedaily, for example once daily or twice daily. A non-limiting example ofdaily administration is a single dose before bedtime.

Also provided herein is a solid pharmaceutical formulation describedherein for reducing food intake. Also provided is a method for reducingfood intake by a subject, by administering to the subject a solidpharmaceutical formulation described herein.

Also provided herein is a solid pharmaceutical formulation describedherein for enhancing insulin secretion. Also provided is a method forenhancing insulin secretion by administering to a subject a solidpharmaceutical formulation described herein. In certain embodiments, thepharmaceutical composition is administered to the subject one or moretimes per day, depending on blood sugar levels. In other embodiments,the pharmaceutical composition is administered to the subject for anextended time.

Also provided herein is a solid pharmaceutical formulation describedherein for prophylactically reducing the incidence of hyperglycemia.Also provided is a method for prophylactically reducing hyperglycemia byadministering to a subject a solid pharmaceutical formulation describedherein. In certain embodiments, the pharmaceutical composition isadministered to the subject one or more times per day, depending onblood sugar levels. In other embodiments, the pharmaceutical compositionis administered to the subject for an extended time.

Also provided herein is a solid pharmaceutical formulation describedherein for prophylactically reducing glucagon secretion. Also providedis a method for prophylactically reducing glucagon secretion byadministering to a subject a solid pharmaceutical formulation describedherein.

Also provided herein is a pharmaceutical formulation having a particularw/w ratio of BBI to KTI in the pharmaceutical composition, comprising,in various embodiments, insulin, a GLP-1 analogue, or a combinationthereof as the active ingredient, for treating diabetes mellitus (DM),which is in some embodiments Type I DM, and is in other embodiments TypeII DM. Also provided is a method for treating DM by administering to asubject a pharmaceutical formulation with a particular BBI: KTI w/wratio. In certain embodiments, the pharmaceutical composition isadministered to the subject one or more times per day, depending onblood sugar levels. In other embodiments, the pharmaceutical compositionis administered to the subject for an extended time. The pharmaceuticalcomposition is, in various embodiments, a liquid pharmaceuticalformulation described herein or a solid pharmaceutical formulationdescribed herein. The aforementioned w/w ratio of BBI to KTI is, in someembodiments, between 1.5:1 and 2.5:1, in other embodiments between 1.6:1and 2.4:1, in other embodiments between 1.7:1 and 2.3:1, in otherembodiments between 1.8:1 and 2.2:1, in other embodiments between 1.9:1and 2.1:1, in other embodiments between 1.95:1 and 2.05:1, in otherembodiments 2:1.

In other embodiments is provided a pharmaceutical formulation having aparticular BBI:KTI w/w ratio, comprising, in various embodiments,insulin, a GLP-1 analogue, or a combination thereof as the activeingredient, for inhibiting or reducing the development of (T2DM) in asubject at risk thereof. In certain embodiments, the subject at risk isconsidered “pre-diabetic”, exhibits IGT, exhibits obesity, and/or has afamily history of diabetes. In certain embodiments, the pharmaceuticalcomposition is administered for an extended time. In more specificembodiments, the pharmaceutical composition may be administered for anextended time daily, for example once daily or twice daily. Anon-limiting example of daily administration is a single dose beforebedtime. The aforementioned w/w ratio of BBI to KTI is, in someembodiments, between 1.5:1 and 2.5:1, in other embodiments between 1.6:1and 2.4:1, in other embodiments between 1.7:1 and 2.3:1, in otherembodiments between 1.8:1 and 2.2:1, in other embodiments between 1.9:1and 2.1:1, in other embodiments between 1.95:1 and 2.05:1, in otherembodiments 2:1.

Coatings

In certain embodiments, the described liquid formulation is containedwithin a capsule, for example a gelatin capsule, which is more specificembodiments itself coated with a pH-sensitive coating. In otherembodiments, a solid formulation, for example, may be coated directlywith a pH-sensitive coating. Those of skill in the art will appreciate,given the present disclosure, that various pH-sensitive coatings may beutilized in the described methods and compositions. In certainembodiments, any coating that inhibits digestion of the composition inthe stomach of a subject may be utilized. Typically, such coatings willnot dissolve in human gastric juices within 2 hours, and will dissolvewithin 30 minutes in duodenal fluid.

In other embodiments, the coating comprises a biodegradablepolysaccharide. In other embodiments, a hydrogel is utilized. In otherembodiments, the coating comprises one of the following excipients:chitosan, an aquacoat ECD coating, an azo-crosslinked polymer, celluloseacetate phthalate, cellulose acetate trimellitate (CAT), celluloseacetate butyrate, hydroxypropylmethyl cellulose phthalate, or poly vinylacetate phthalate.

In other embodiments, a timed-release system such as Pulsincap™ isutilized.

In preferred embodiments, the coated dosage forms described hereinrelease the core (containing the oil-based formulation) when pH reachesthe range found in the intestines, which is alkaline relative to that inthe stomach. In more specific embodiments, the coating comprises apH-sensitive polymer. In various embodiments, either mono-layer ormulti-layer coatings may be utilized.

In one embodiment, the coating is an enteric coating. Methods forenteric coating are well known in the art (see, for example, Siepmann Fet al 2005). In more specific embodiments, a Eudragit™ coating isutilized as the enteric coating. Eudragit™ coatings are acrylicpolymers, the use of which is well known in the art.

In another embodiment, microencapsulation is used as a stomach-resistantcoating in the compositions described herein. Methods formicroencapsulation are well known in the art, and are described interalia in U.S. Patent Application Publication No. 2011/0305768, which isincorporated by reference herein.

In other embodiments, the described pharmaceutical composition is in theform of a capsule. Gelatin capsules are most preferred, which may besoft gelatin capsules, or in other embodiments hard gelatin capsules.Methods for inserting an oil-based formulation into a gelatin capsuleare well known in the art. In more specific embodiments, the gelatincapsule is itself coated by an enteric coating.

A variety of oral dosage forms, such as tablets, capsules, pills,powders, and granules, may be used in the described solid compositions.Suitable excipients can be chosen among, but are not restricted to,solid powdered carriers, e.g. US Pharmacopeia and the Handbook ofPharmaceutical Excipients, for example mannitol, microcrystallinecellulose, calcium hydrogen phosphate, calcium sulphate, and starch;binders, e.g., polyvinylpyrrolidone, starch and hydroxypropylmethylcellulose; disintegrants, e.g., sodium croscarmellose, sodiumstarch glycollate and polyvinylpyrrolidone as well as lubricatingagents, e.g., magnesium stearate, sodium stearyl fumarate, talc andhydrogenated vegetable oil such as Sterotex NF. In some embodiments,lactose-free compositions contain active ingredients, a binder/filler,and a lubricant in compatible amounts. More specific embodiments oflactose-free dosage forms contain active ingredients, microcrystallinecellulose, pre-gelatinized starch, and magnesium stearate. The mixtureis then processed, in certain embodiments, into tablets or granules forcapsules. In certain embodiments, rapid disintegrating tablets areutilized, which are in some embodiments useful for elderly or infirmindividuals who have difficulty swallowing. In other embodiments, theoral dosage form is enteric-coated.

Wherever alternatives for single separable features such as, forexample, an insulin protein or dosage thereof, a GLP-1 analogue ordosage thereof, a protease inhibitor or amount thereof, a chelator oramount thereof, an emulsifier or amount thereof, a non-ionic detergentor an amount thereof, or a capsule and/or coating are set forth hereinas “embodiments”, it is to be understood that such alternatives may becombined freely to form discrete embodiments of the invention disclosedherein.

With respect to the jurisdictions allowing it, all patents, patentapplications, and publications mentioned herein, both supra and infra,are incorporated herein by reference.

The invention is further illustrated by the following examples and thefigures, from which further embodiments and advantages can be drawn.These examples are meant to illustrate the invention but not to limitits scope.

EXPERIMENTAL DETAILS SECTION

In all the animal experiments described herein, liquid dosage forms thatare administered to a human in an enteric-coated capsule may, forexample, be administered directly to the digestive system of the animalvia a cannula. In general, liquid and solid dosage forms may be fed toanimals via gavage.

Example 1: Testing of Oral GLP-1 Analogue and/or Insulin Formulationsfor Treating NAFLD

Volunteers with NAFLD are administered one or more dosage forms having apH-sensitive coating and/or capsule containing one or more proteaseinhibitors; EDTA; and a GLP-1 analogue, on an ongoing basis, for examplefor a time period of between 1-24 months. In other experiments, thedosage form contains insulin and a GLP-1 analogue. In still otherexperiments, subjects at risk of developing NAFLD are treated with thecomposition. The NAFLD status of the subjects is followed over theexperimental period, to test the effectiveness of the compositions.

Example 2: Testing of Oral GLP-1 Analogue and/or Insulin Formulationsfor Other Metabolic Indications

Volunteers with metabolic disorder (Grundy et al, 2004) are administeredone or more dosage forms having a pH-sensitive coating and/or capsuleand containing one or more protease inhibitors; EDTA; and a GLP-1analogue, on an ongoing basis, for example for a time period of 1-24months. In other experiments, the dosage form contains insulin and aGLP-1 analogue. In still other experiments, subjects at risk ofdeveloping metabolic disorder are treated with the composition. Subjectsare followed over the experimental period for obesity (for example bymeasuring waist circumference), total cholesterol levels,hypertriglyceridemia, serum ApoB levels, total cholesterol/HDL ratios,ApoB/ApoA1 ratios, atherosclerosis, sub-clinical inflammation (as can bemeasured inter alia by measuring levels of C-reactive protein), thepresence of a prothrombotic state (as can be measured inter alia bymeasuring levels of plasminogen activator inhibitor-1 [PAI-1]), thepresence of platelet activation, the presence of endothelialdysfunction, the presence of a cardioembolic state, and/orinsulin-induced enhancement of vasodilator responses (Sung et al, 2012;Chatrath et al 2012; Nseir et al, 2011).

Example 3: Testing of Oral GLP-1 Analogue Formulations for Treating andPreventing Alzheimer's Disease in an Animal Model

One or more dosage forms containing one or more protease inhibitors,EDTA, and a GLP-1 analogue is administered on an ongoing basis, forexample for a time period of between 1-24 weeks, to experimental animalsin the context of an animal model of Alzheimer's disease. In otherexperiments, the formulation contains insulin and a GLP-1 analogue asthe active agents. In some experiments, the animal model is astreptozotocin (STZ)-induced rat model of AD. Intracerebral injection ofSTZ leads to hyper-phosphorylation of tau protein and causes a conditionthat mimics AD. Some experiments utilize sham-injected animals, wherebyCSF is used instead of STZ, as a control group.

After complete recovery from the procedure (typically several monthsafter induction), animals are divided into groups and for treatment withoral GLP-1 or empty carrier (e.g. normal saline) for a period of severaldays, in some experiments for at least 30 days.

Dose-dependent and time-course effects of oral GLP-1 on memory retentionare measured during the course of treatment. Following treatment, theanimals are sacrificed, and brain tissues are used to evaluatehippocampal and cortical GLP-1 levels, amyloid beta (Aβ) burden, tauphosphorylation, and inflammatory markers.

In some experiments, the cognitive status of the subjects is followedover the experimental period, or neurodegenerative disease status isassessed according to Salcedo et al or a reference cited therein, totest the effectiveness of the compositions. Below are somerepresentative protocols that can be used:

Radial arm maze (RAM) task

Working memory in animals is tested with the RAM apparatus (Alamed) andusing described test methods including training. The test is typicallyperformed several months after STZ induction.

Hole-board (HB) task

Animals are tested for learning deficits in a food-motivated complex HBapparatus. The apparatus consists of an open field containing an arrayof holes surrounded by Plexiglass walls. Each hole contains a metal cupwith a perforated bottom under which food pellets are placed. The testrecords the number of visits to food-baited holes (hits), the number ofvisits to unbaited holes (errors) and the time to complete the trial.

Biochemical studies

Animals are euthanized at the completion of behavioral studies, andbiochemical studies and histology are conducted on fixed brains. In someexperiments, the hippocampus and frontal cortex from one hemisphere aredissected and used for biochemical studies, such as those listed below.

Estimation of active GLP-1 levels

Isolated hippocampal and cortex samples are homogenized with a 10-foldvolume of chilled 50 mM phosphate-buffered saline (pH 7.8). Thehomogenate is divided into four equal portions and utilized for theestimation of GLP-1, Aβ42, tau and inflammatory markers. Active GLP-1and Aβ42 levels can be tested using commercially available ELISA kits.

Measurement of phosphorylated tau (p-tau)

Hippocampal and cortical tissues are harvested and subjected to WesternBlot analysis.

Measurement of TNF-α and IL-1β levels

TNF-α and IL-1β levels in the hippocampal and cortex homogenate can bemeasured using available ELISA kits.

Histology and neuronal count

The hippocampus and cortex is sectioned and stained with cresyl violet(CV) acetate, and stained neurons are analyzed using an image analyzer.CV positive neurons numbers are compared with the sham control group,and average cell counts of the sections are obtained from each animal.

Example 4: Testing of Oral GLP-1 Analogue Formulations for Treating andPreventing Stroke In An Animal Model

The preventive and therapeutic effects of oral GLP-1 analogue are testedin an animal stroke model. One such model is a reperfusion injury modelin rats, where rats undergo temporary occlusion of the middle cerebralartery for 90 min. Rats are administered GLP-1 analogue or carrier forseveral days prior and/or after reperfusion. Assessment of theneurological consequences, biochemical changes, and/or size of infarctis performed. Neurological function may be determined using a modifiedBederson's test at one or several time points after occlusion, afterwhich rats may be euthanized for histological investigation. In someexperiments, peripheral blood is obtained for measurement of bloodglucose level and evaluation of oxidative stress and/or brain tissuesare collected to measure vascular endothelial growth factor (VEGF)levels (Sato et al).

Example 5: Testing of Oral GLP-1 Analogue Formulations for Treating andPreventing Parkinson'S Disease

Patients with moderate Parkinson's disease (PD) are randomly assigned toreceive either oral GLP-1 analogue or placebo for 12 months. PDprogression is measured, in some cases after withdrawal of conventionalPD medication. For example, the Movement Disorders Society UnifiedParkinson's Disease Rating Scale (MDS-UPDRS) may be used, in some casestogether with one or more non-motor tests, at several time points duringtreatment and in some cases after a further washout period (Aviles-Olmoset al).

Example 6: Testing of Oral GLP-1 Analogue Formulations for Treating andPreventing Traumatic Brain Injury (TBI) in an Animal Model

The ability of oral GLP-1 analogue formulations to protect against orfacilitate recovery from TBI is evaluated. In some experiments, theGLP-1 analogue is administered for several days after injury. One modelthat may be used is an in vivo fluid percussion injury model (Eakin etal). Markers of cell death and measures of cognitive function may beutilized. Examples of the latter are the Morris Water Maze and othercognitive tests described herein.

Example 7: Testing of Oral GLP-1 Analogue Formulations for Treating andPreventing Peripheral Nerve Injury in an Animal Model

The ability of oral GLP-1 analogue formulations to protect against orfacilitate recovery from peripheral nerve disease is evaluated. Onemodel that may be used is sciatic nerve crush nerve injury. GLP-1analogue or empty carrier is administered shortly after crush injury andcontinued for several subsequent days or weeks. Rats subjected tosciatic nerve crush may exhibit marked functional loss,electrophysiological dysfunction, and atrophy of the tibialis anteriormuscle (TA). Recovery can be monitored by measuring neurologicalfunction, electrophysiological function, muscle atrophy, and/ormorphological parameters several days or weeks after nerve crush(Yamamoto et al).

Example 8: Testing of Oral GLP-1 Analogue Formulations for Treating andPreventing Cognition and Mood Disorders

In other experiments, the ability of oral GLP-1 analogue formulations totreat bipolar disorder, major depressive disorder, schizophrenia, and/orschizoaffective disorder is evaluated. Individuals with these conditionsor at risk of developing them are treated with GLP-1 analogue on anongoing basis, for example for a time period of between 1-24 months, andclinical global improvement in psychiatric symptoms and/or manic,depressive, or schizophrenic symptoms is measured. In other experiments,cognitive function is determined, using tests known to those skilled inthe art (McIntyre et al).

Example 9: Testing of Oral GLP-1 Analogue Formulations for Treating andPreventing Amyotrophic Lateral Sclerosis (ALS) in an Animal Model

In other experiments, the ability of oral GLP-1 analogue formulations totreat or prevent ALS is evaluated. GLP-1 analogue is administered on anongoing basis, for example for between 1-18 weeks. In some experiments,an animal model such as the SOD1 G93A mutant mouse (Li et al) isutilized. Disease progression can be monitored by measuring activitylevel, e.g. running behavior, lumbar spinal cord structure, and, usingbrain tissue, neuron density and specific disease progression markerssuch as glial fribrillary acidic protein (GFAP), Caspase-3, Cholineacetyl transferase (ChAT), and the neuronal cell neurofilament protein,SMI-32 (Li et al).

Example 10: Testing of Oral GLP-1 Analogue Formulations for Treating andPreventing Huntington's Disease in an Animal Model

In other experiments, the ability of oral GLP-1 analogue formulations totreat or prevent Huntington's disease is evaluated. GLP-1 analogue isadministered on an ongoing basis, for example for between 1-18 weeks. Insome experiments, an animal model such as those described in Brooks andDunnett and the references cited therein is utilized.

Example 11: Testing of Oral GLP-1 Analogue Formulations for Treating andPreventing Diabetic Neuropathy in an Animal Model

In other experiments, the ability of oral GLP-1 analogue formulations totreat or prevent diabetic neuropathy is evaluated. GLP-1 analogue isadministered on an ongoing basis, for example for between 1-18 weeks. Insome experiments, an animal model such as those described in Lai and Loand the references cited therein is utilized.

Example 12: Testing of Oral GLP-1 Analogue Formulations for Treating andPreventing Alzheimer's Disease, Huntington's Disease, Stroke, TBI,Peripheral Nerve Injury, AL, And Diabetic Neuropathy in Humans

One or more dosage forms containing one or more protease inhibitors,EDTA, and a GLP-1 analogue is administered on an ongoing basis, forexample for between 1-48 months, to subjects at risk of developingAlzheimer's disease, Huntington's disease stroke, TBI, peripheral nerveinjury, ALS, or diabetic neuropathy. In other experiments, theformulation contains insulin and a GLP-1 analogue. To test theeffectiveness of the compositions, the disease status of the subjects isfollowed over the experimental period, in some cases in combination withbrain imaging. Disease status may be followed, for example, byperforming physiological tests and/or determining cognitive status orneurological function, according to Salcedo et al or a reference citedtherein, or other tests known to those skilled in the art.

Example 13: Testing of Oral GLP-1 Analogue Formulations for Treating andPreventing Diabetic Neuropathy in an Animal Model

In other experiments, the ability of solid pharmaceutical formulations,containing insulin, a GLP-1 analogue, or a combination thereof, toinhibit or prevent the development of Type II Diabetes Mellitus (T2DM)in a subject with impaired glucose tolerance (IGT) is evaluated. Thepharmaceutical composition is administered on an ongoing basis, forexample for between 1-240 weeks, in some experiments daily at bedtime.Patients are followed by DM indicia known in the art, such as fastingplasma glucose (FPG) levels, frequently sampled intravenous glucosetolerance test (FSIVGTT), oral glucose tolerance test (OGTT), and HbA1Clevels, to monitor the development of T2DM. Alternatively or inaddition, insulin sensitivity and beta-cell function are measured.

In the claims, the word “comprise”, and variations thereof such as“comprises”, “comprising”, and the like indicate that the componentslisted are included, but not generally to the exclusion of othercomponents.

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1. An oral pharmaceutical composition for inhibiting the development ofor treating nonalcoholic fatty liver disease (NAFLD), saidpharmaceutical composition comprising an insulin, a GLP-1 analogue, or acombination thereof; a protease inhibitor; and a chelator of divalentcations.
 2. The oral pharmaceutical composition of claim 1, where saidpharmaceutical composition is administered for more than one month. 3.The oral pharmaceutical composition of claim 1, where saidpharmaceutical composition is administered once daily or twice daily. 4.The oral pharmaceutical composition of claim 1, where saidpharmaceutical composition comprises a liquid formulation; where saidinsulin, GLP-1 analogue, or combination thereof, said proteaseinhibitor, and said chelator of divalent cations are in said liquidformulation.
 5. The oral pharmaceutical composition of claim 4, wheresaid liquid formulation is inside a capsule.
 6. The oral pharmaceuticalcomposition of claim 5, where said capsule is surrounded by a coatingthat resists degradation in the stomach.
 7. The oral pharmaceuticalcomposition of claim 4, where said liquid formulation is an oil-basedliquid formulation.
 8. The oral pharmaceutical composition of claim 1,where said pharmaceutical composition is a solid formulation.
 9. Theoral pharmaceutical composition of any of claims 1-8, where said GLP-1analogue is present in an amount between 100-600 micrograms (inclusive)per dose for an adult patent or a corresponding amount per body weightfor a pediatric patient.
 10. The oral pharmaceutical composition ofclaim 9, where said GLP-1 analogue is present in an amount between100-300 micrograms (inclusive) per dose for an adult patent or acorresponding amount per body weight for a pediatric patient.
 11. Theoral pharmaceutical composition of claim 9 or 10, where said GLP-1analogue is exenatide.
 12. The oral pharmaceutical composition of any ofclaims 1-8, where said insulin is present in an amount between 8-32 mg(inclusive) per dose for an adult patent or a corresponding amount perbody weight for a pediatric patient.
 13. The oral pharmaceuticalcomposition of any of claims 1-8, where said insulin is present in anamount between 4-12 mg (inclusive) per dose for an adult patent or acorresponding amount per body weight for a pediatric patient.
 14. Theoral pharmaceutical composition of claim 13, where, in addition to saidinsulin, said GLP-1 analogue is present in an amount between 100-300micrograms (inclusive) per dose for an adult patent or a correspondingamount per body weight for a pediatric patient.
 15. The oralpharmaceutical composition of claim 14, where said GLP-1 analogue isexenatide.
 16. The oral pharmaceutical composition of any of claims1-15, where said protease inhibitor is selected from the groupconsisting of soybean trypsin inhibitor (SBTI), Bowman-Birk inhibitor(BBI), Kunitz Trypsin Inhibitor 3 (KTI3), and aprotinin, each of whichmay be present either alone or in combination with another proteaseinhibitor.
 17. The oral pharmaceutical composition of any of claims1-15, where one or more protease inhibitors are present, and saidprotease inhibitors collectively inhibit both trypsin and chymotrypsin.18. The oral pharmaceutical composition of any of claims 1-15, where twoprotease inhibitors are present in said composition, and said twoprotease inhibitors are SBTI and aprotinin.
 19. The oral pharmaceuticalcomposition of any of claims 1-15, where two protease inhibitors arepresent in said composition, and said two protease inhibitors areisolated KTI3 and isolated BBI.
 20. The oral pharmaceutical compositionof any of claims 1-15, where two protease inhibitors are present in saidcomposition, and said two protease inhibitors are (i) isolated KTI3 and(ii) aprotinin.
 21. The oral pharmaceutical composition of any of claims1-15, where one protease inhibitor is present in said composition, andsaid protease inhibitor is isolated BBI.
 22. The oral pharmaceuticalcomposition of any of claims 1-21, where said chelator is EDTA.
 23. Theoral pharmaceutical composition of any of claims 7-22, where said oil isselected from olive oil, flaxseed oil, sesame oil, avocado oil, walnutoil, canola oil, and fish oil.
 24. The oral pharmaceutical compositionof claim 23, where said oil is fish oil.
 25. The oral pharmaceuticalcomposition of any of claims 7-24, where said oil-based liquidformulation is water-free.
 26. The oral pharmaceutical composition ofany of claims 1-10, 12-14, and 16-25, where said GLP-1 analogue isexenatide.
 27. A method for inhibiting the development of or treatingNAFLD in a human subject, said method comprising the step ofadministering said subject the oral pharmaceutical composition of any ofclaims 1-26, thereby inhibiting the development of or treating NAFLD.28. The method of claim 27, where said pharmaceutical composition isadministered for more than one month.
 29. A method for inhibiting thedevelopment of or treating NAFLD in a non-human subject, said methodcomprising the step of administering to said non-human animal the oralpharmaceutical composition of any one of claims 1-26, thereby inhibitingthe development of or treating NAFLD.
 30. The method of claim 29, wheresaid pharmaceutical composition is administered for more than one month.